Introduction: Controversies about effects of the forces formed during root canal treatment on dentinal microcracks formation still exist due to different results reported. The issue of methodology, sample selection and storage of the specimens still require further analysis, particularly because new instruments are constantly being developed. The aim of the study was to explore microcrack formation after root canal preparation with Self-adjusting File (SAF), Reciproc Blue (RB), and ProTaper Next (PTN) instruments on young premolars by means of micro computed tomography. Methods: Fifty-seven premolars, two-rooted with one canal (Vertucci type I) or single-rooted teeth of Vertucci type II had been used for the study All the teeth were visually inspected under the stereomicroscope with magnification of 12 x to exclude those with open apices, those that were root fractured during the extractions and pre-existing dentinal defects. The specimens were decoronated using a diamond disc to standardize the working length about 12 mm. The periodontal ligament was simulated by a thin layer of polysiloxane impression material on the root surface and the samples were embedded in blocks made of acrylic resin. Specimens were scanned at geometrical magnification of 6.3, which yielded a structural resolution of 20.2 μm. All teeth were scanned at X-ray energy of 110 kV and X-ray tube current of 140 μA with a 0.1 mm Cu filter, using 1440 projections at an exposure time of 333 ms. Beam hardening was reduced using a Hanning filter. Noise was reduced using a median filter, and surface detection was performed using an adaptive search algorithm (Volume Graphics VGStudio Max 3). Samples were randomly assigned into three groups of 19 teeth each according the instrumentation technique: Self-Adjusting File (SAF), Reciproc Blue (RB), and ProTaper Next (PTN) For the SAF group (vibration kinematics), the glide path was created using the Pre-SAF set of instruments (ReDent Nova). An RDT3 handpiece was used for canal preparation with SAF instrument 1.5 mm in diameter at frequency of 5000 vibrations per minute and amplitude of 0.4 mm for a total of four minutes. The canals were simultaneously irrigated with 2.5% NaOCl at a flow rate of 4 ml/min for three minutes and one minute with 4 ml of 17% ethylenediaminetetraacetic acid (EDTA) by use of a syringe and needle. In the RB group (reciprocation kinemamatics), glide path was created with R-Pilot (WDV, Munich, Germany) followed with Reciproc Blue until the working length was reached The glide path in the PTN group (full rotary kinematics) was created with Proglider (Dentsplay Maillefer) ProTaper Next X1 and subsequently ProTaper Next X2 were each used in a brushing motion at a speed of 300 rpm with torque set to 4 Ncm to progress apically until reaching the working length. During instrumentation in RB and PTN group, each canal was irrigated with 12 ml of 2.5% NaOCl, and final irrigation was performed with 4 ml of 17% EDTA to match the irrigation protocol used in SAF grous. After the root canal preparation, post-operative scans were performed in the same specimen positions and with the same micro-CT parameters as the first scan. In order to check the influence of the moisture on visibility of microcracks, specimens were kept in dry conditions for 24 hours and then scanned for the third time. Two calibrated, blinded examiners evaluated the cross-sectional images using the imageprocessing software Volume Graphics VGStudio Max 3. The roots were divided into cervical, middle, and apical parts. Radiolucent lines present in the micro-CT sections would be categorized as microcracks. If the microcracks had been observed on post-operative scans, the corresponding pre-operative scan would have been analyzed to exclude pre-existing defects. Ten specimens of each group were cut in horizontal slices and tested on fracture resistance in a universal testing machine AGS-X, Shimadzu (Japan) at speed 0,5 mm/min. Results: No dentinal defect was found in any evaluated specimen, neither in pre- and nor postoperative scans in wet and dry condition. PTN group showed the lowest value for fracture resistance (419,15 N) compering to RB (485,57 N) and (473,12 N). The differences were not statistically significant (p=0.151). Conclusion: Under the circumstances of this study instruments with three different kinematics (SAF, Reciproc Blue and Protaper Next) do not cause dentinal microcracks in young premolar teeth and does not cause differences in resistance to load to fracture.