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Background: Fecal calprotectin (FCP) is a highly sensitive biomarker of intestinal inflammation widely used in diagnostics and monitoring of inflammatory bowel disease (IBD). Immunohistochemical assessment of calprotectin in the bowel mucosa is not a diagnostic standard. Therefore, the aim of this study was to evaluate tissue calprotectin (TCP) as a potential marker providing added insight for pediatric patients with Crohn’s disease (CD). Methods: Fecal and tissue calprotectin were measured in children with CD. The values were correlated with disease activity and histopathological changes of the patients’ endoscopic biopsies. Disease activity was assessed using the Pediatric Crohn’s Disease Activity Index (PCDAI); fecal calprotectin (FCP) was measured with the ELISA test. Immunohistochemical (IHC) staining for calprotectin antigen was performed on the biopsy samples from six bowel segments, and the number of TCP cells was counted per high power field (HPF). Non-parametric statistical tests were used for data analysis. Results: Fifty-seven children with CD with a median age of 10.5 (1–17) years (yrs) were examined for fecal and tissue calprotectin. The patients’ median PCDAI score was 10 (0–63.5), while median FCP was 535 (30–600) μg/g. We observed a correlation between disease activity (PCDAI) and FCP, TCP in inflammatory lesions and in crypts. There was no association either between FCP and TCP or between TCP in epithelium and PCDAI. Conclusion: It seems that IHC detection of calprotectin in bowel mucosa to assess disease behavior may be useful. FCP is a gold-standard biomarker in the diagnosis, monitoring and prognosis of IBD, and its levels correlated well with clinical activity in our study group.