Twenty-four cases of primary nonampullary small intestinal adenocarcinoma were immunohistochemically examined for the expression of cytokeratin (CK) 7 and CK20 and compared with 23 colorectal adenocarcinomas secondarily involving the small intestine by direct extension or metastasis. While normal small intestinal mucosa was diffusely positive for CK20 and completely negative for CK7 expression, all small intestinal adenocarcinomas (24 of 24) showed a variable degree of CK7 expression. Specifically, the CK7 staining pattern was diffuse in 13 cases (54%) and focal in the remaining cases. Sixteen small intestinal adenocarcinomas (67%) coexpressed CK7 and CK20, and 8 (33%) completely lost CK20 immunoreactivity when compared with adjacent non-neoplastic small intestinal mucosa. In the latter cases, the loss of CK20 immunoreactivity with a reciprocal emergence of CK7 expression was evident. This was in contrast to secondary colorectal adenocarcinomas where 22 cases (96%) expressed CK20, among which only 1 case showed focal CK7 expression. The remaining 1 case was negative for both CK7 and CK20. Interestingly, adenomatous epithelium associated with small intestinal adenocarcinomas identified in 18 cases also exhibited CK7 positivity with a sharp transition from CK7-negative normal-appearing epithelium. Taken together, these observations delineate an alteration of CK7 and CK20 expression profile that occurs early in small intestinal tumorigenesis. This unique pattern may be of diagnostic value in distinguishing primary small intestinal adenocarcinoma from secondary colorectal adenocarcinoma.