INTRODUCTION: The study was conducted to examine and compare the diagnostic values of standard methods of histological analysis (hematoxylin and eosin staining, histochemical staining for acetylcholinesterase) with immunohistochemical staining for calretinin of rectal biopsies obtained by rectal suction biopsy in patients with suspected Hirschsprung’s disease. ----- METHODS: A retrospective analysis of the medical records of all children who underwent a rectal suction biopsy from January 2016 to May 2021 at the University Hospital Center Zagreb due to suspected Hirschsprung's disease. The basic characteristics of the patient and the findings of diagnostic and therapeutic procedures were extracted. Specificity, sensitivity, positive predictive value, and negative predictive value were calculated to compare diagnostic tests. ----- RESULTS: Data from 24 participants were analyzed. The diagnosis of Hirschsprung's disease was confirmed in seven (29%) patients. The most common reasons for suspicion of Hirschsprung's disease and indications for performing a rectal suction biopsy were abdominal distension (66.7%), constipation (62.5%) and vomiting (41.7%). In the diagnosis of Hirschsprung's disease, immunohistochemical staining for calretinin showed the highest specificity (85%) and sensitivity (100%), and histochemical staining of acetylcholinesterase showed the lowest specificity (66.7%) and sensitivity (37.5%). ----- CONCLUSION: Immunohistochemical staining of calretinin has high diagnostic accuracy. The introduction of immunohistochemical staining for calretinin in the standard diagnostic procedure could accelerate and improve the accuracy of the diagnostic procedure by eliminating difficulties that often complicate the timely diagnosis of Hirschsprung's disease.