The tyrosine-specific protein kinase activity of pp60c-src molecules obtained from RIPA buffer lysates of human colon tumor-derived cell lines is elevated over that found in lysates from normal human colon mucosa cells. The elevation of pp60c-src kinase activity in the lysates of the cultured colon tumor cells does not appear to be the result of pp60c-src overexpression suggesting that the specific activity of the pp60c-src phosphotransferase may be enhanced. Cell-free translation of c-src mRNA from colon tumor cells and normal colon mucosa cells yielded pp60c-src molecules with similar levels of in vitro protein kinase activity suggesting that pp60c-src kinase activity in these cells may be regulated differently at a post-translational level. Analysis of pp60c-src molecules from normal colon and colon carcinoma cells revealed that they possessed indistinguishable sites and quantities of phosphorylated serine and tyrosine residues and were not detectably complexed with other cellular proteins. The activation of pp60c-src kinase activity in the colon tumor cells is associated with an apparent increase in the turnover rate of tyrosine-phosphates within the carboxyl terminal portion of the pp60c-src molecules from these tumor derived cell lines.