Background and Aims: Fecal calprotectin [fcal] is a biomarker of Crohn's disease [CD] endoscopic activity. Identifying the endoscopic situations in which fcal is less reliable remains unexplored. We aimed to determine the endoscopic factors influencing fcal level in CD. Methods: Overall, 53 CD patients consecutively and prospectively underwent colonoscopy, with CD Endoscopic Index of Severity [CDEIS] calculation and stool collection. Fcal was measured using a quantitative immunochromatographic test. Correlation analysis was done with Pearson statistics. Results: Fcal was correlated with CDEIS [0.66, p < 0.001]. In univariate analysis, fcal was correlated with the affected surface [0.65, p < 0.001] and the ulcerated surface [0.47, p < 0.001]. Fcal was significantly associated with ulceration depth, with median fcal of 867.5 mu g/g, 1251.0 mu g/g, and 1800.0 mu g/g, in patients presenting with non-ulcerated lesions, superficial ulcerations [SU], and deep ulcerations [DU], respectively. Lesion locations did not influence fcal. In multivariate analysis, fcal was associated with affected surface [p = 0.04] and the presence of CD lesions. Moreover, fcal increased with the ulceration depth [p = 0.03]. However, ulcerated surface and CD location did not affect fcal. Using a receiver operating characteristic [ROC] curve, we showed that fcal of 400 mu g/g was the best compromise between sensitivity [0.76] and specificity [0.77], whereas fcal >= 200 mu g/g was highly sensitive [0.86] to detect SU or DU. Conclusions: Fcal is a very reliable biomarker to detect endoscopic ulcerations in CD. We suggest repeating measurement in case of intermediary results [200-400 mu g/g] in daily practice. Fcal level is mostly influenced by the presence of CD lesions [even non-ulcerated], in a depth-related manner and by the affected surface.