Recent advances in endocytoscopy have enabled in vivo evaluation not on ly of structural atypia, but also of cellular atypia with observation of lumens and nuclei in the surface layer of the mucosa. The aim of this prospective pilot study was to evaluate the usefulness of our novel endocytoscopic classification in colorectal lesions. A total of 206 consecutive patients were enrolled in the study and underwent endocytoscopic examination. Endocytoscopic images were stored electronically and two endoscopists blinded to the findings at live examination assigned them diagnoses using the endocytoscopic (EC) classification. The endocytoscopic diagnosis was then compared to the final histopathological diagnosis. In all, 196 patients with 213 specimens were available for analysis. All normal mucosae were classified as EC1a and all hyperplastic polyps as EC1b. Dysplasias were mainly classified as EC2, while massively invasive submucosal cancers (SMm) or worse, which have the possibility of metastasis, were mainly EC3b. Assuming that an EC1b classification was diagnostic of hyperplastic polyps, we were able to differentiate nonneoplastic from neoplastic lesions with a sensitivity of 100 % and a specificity of 100 % (P < 0.05). Assuming that an EC3b classification was diagnostic of SMm or worse, we were able to differentiate "SMm or worse" from other neoplastic lesions (dysplasias and slightly invasive submucosal cancers) with a sensitivity of 90.1 % and a specificity of 99.2 % (P < 0.05). The endocytoscopic classification was particularly useful for differentiating between neoplastic and nonneoplastic lesions and between "SMm or worse" and other neoplastic lesions, which in the case of colorectal neoplasms would help to determine treatment.