To examine the role of Src-related proteins in human colon carcinoma we measured the tyrosine kinase activity of pp60c-src (Src), p62c-yes (Yes), p56lck (Lck), p59fyn (Fyn), p59hck (Hck), p56lyn (Lyn) and p55c-fgr (Fgr) from colonic cells. Yes activity, similar to that of Src, was 10-20 fold higher in three of five colon carcinoma cell lines and fivefold higher in 10 of 21 primary colon cancers than that in normal colonic cells. Lck activity was present in COLO 205 cells, otherwise Lck, Fyn, Hck, Lyn and Fgr activities were not detected in any of the carcinoma cell lines or cancers tested. Increased Yes activity, like that of Src, was due mostly to increased protein levels and not to an apparent decrease in phosphorylation of Tyr 537, the major mechanisms known to deregulate enzymatic activity. Only those colon carcinoma cell lines with elevated Src and/or Yes tyrosine kinase activity as measured in vitro had elevated levels of three tyrosine-phosphorylated proteins as measured in vivo. Thus, colon carcinoma cells contain active tyrosine kinases and/or inactive tyrosine phosphatases not present in normal colonic cells, and Src and Yes appear to be active kinases in the carcinoma cells. These data, together with those demonstrating decreased Src activity in fully differentiated enterocytes, suggest that down regulation of Src-related tyrosine kinases is important for differentiation, and/or deregulation of the kinases is important for growth and transformation of intestinal epithelial cells.