Aims: To investigate the expression of metallothioneins (MTs) and free zinc content in gallbladderassociated with cholecystolithiasis. Study Design: Gallbladder tissue samples were obtained from patients, subjected to laparoscopic cholecystectomy, owing to the cholecystolithiasis, verified by hematological examination and by ultrasonography. Operations were performed in clinical stable phase of gallstone disease. Place and Duration of Study: Department of Surgery, Clinical Hospital Center, Departments of Physiology and Immunology and Department of Pathology, Medical Faculty, University of Rijeka, between June 2011 and June 2012. Methodology: In 25 collected gallbladders the degree of inflammation was classified as subacute (SA) or chronic cholecystitis (CC), according to the standard pathohistological analysis and intensity of NF-B expression. The tissue expression of MT-I/II isoforms and free zinc content werevisualized by immunohistochemistry, using the monoclonal anti-MT I+II antibodies and zinc fluorophore-Zinquin. The cell-based staining quantification was performed by Cell F v3.1 software. Results: The data have shown that in 20 patients out of 25 patients the gallstones induced the CC with upregulation of NF-B in gallbladder epithelium, as well as that high inflammation was followed by overexpression MTs and accumulation of Zn. in 15 and 12 out of 20 patients, respectively. Furthermore, histomorphometric analysis revealed that the average grey values for MT I/II in CC arose from 49.8 +/- 3.3 (in SC) to 195.4 +/- 15.5 (in epithelial cells) and from 62.3 +/- 21.0 to 194.8 +/- 20.1 (in stromal cells) (P<0.01). Similarly, average values for zinc ions increased from 25.6+/-7.4 (in SC) to 118.8 +/- 5.7 (in epithelial cells). Besides, in areas of high inflammation several CD3+cells were found in contact with MT+ epithelial, endothelial and monocyte-like cells. Conclusion: The data point to high local activation of MT/zinc network in chronic cholecystitis associated by gallstones, emphasizing its regulatory functions in inflammatory environment.