Small intestinal adenocarcinoma is an uncommon neoplasm morphologically similar to or indistinguishable from colorectal adenocarcinoma. Although much has been learned about genetic pathways critical to colorectal tumorigenesis, little is known about molecular alterations involved in the development of small intestinal adenocarcinoma. In this study, we immunohistochemically compared non-ampullary small intestinal adenocarcinomas with sporadic colorectal adenocarcinomas for the expression of several proteins known to serve pivotal roles in colorectal tumorigenesis. These included adenomatous polyposis coli and beta-catenin involved in the Wnt signaling pathway, and DNA mismatch repair enzymes hMLH1, hMSH2 and hMSH6 involved in the microsatellite instability pathway. The expression of two important tumor suppressors, p53 and RB, was also examined. The results show that complete loss of adenomatous polyposis coli immunoreactivity, presumably resulting from its gene mutations, was observed in eight of 26 (31%) small intestinal adenocarcinomas and 36 of 51 (71%) colorectal adenocarcinomas (P = 0.0008). Nuclear localization of beta-catenin, an indirect evidence of deregulated Wnt signaling pathway, was observed in 5 (19%) small intestinal adenocarcinomas and 36 (71%) colorectal adenocarcinomas (P<0.0001). Total lack of nuclear staining for one or more of the DNA mismatch repair enzymes occurred in a similar low frequency in both small intestinal and colorectal adenocarcinomas, seen in two of 25 (8%) and 10 of 47 (21%) cases, respectively (P = 0.1958). The frequencies of aberrant p53 and RB expression were also similar between small intestinal and colorectal adenocarcinomas. These observations indicate that defects in the Wnt and microsatellite instability pathways occur in over 90% of colorectal adenocarcinomas, but in only 40% of small intestinal adenocarcinomas. Small intestinal tumorigenesis appears to follow a distinct, yet unidentified, molecular pathway(s) from its colorectal counterpart despite their morphologic similarity.