Taking advantage of the potent mitogenic effect exerted by insulin in human colonic cells, we used Caco-2 cells transfected with an activated (Val-12) human Haras gene or the polyoma middle T (PyMT) oncogene, a constitutive activator of pp60c-src tyrosine kinase activity, to investigate the effect of oncogenic p21ras and PyMT/pp60c-src on insulin mitogenic signaling. As compared to vector control Caco-2 cells, both oncogene-transfected cells exhibited: 1) a loss of response to insulin's stimulatory effect on mitogen-activated protein (MAP) kinase activity and cell proliferation, both of which were constitutively increased; 2) a decrease in insulin receptor (IR) affinity and insulin-stimulated exogenous tyrosine kinase activity, which resulted from increased protein kinase C (PKC) activity (Delage, S., Chastre, E., Empereur, S., Wicek, D., Veissiére, D., Capeau, J., Gespach, C., and Cherqui, G. (1993) Cancer Res. 53, 2762-2770), since IR alterations were corrected by PKC down-regulation; and 3) a decrease in both IR mRNA level and IR number, which was independent of PKC since it persisted after PKC down-regulation. In conclusion, this is the first evidence that oncogenic p21ras and PyMT/pp60c-src abolish insulin mitogenic signaling in human colonic cells through mechanisms involving (i) constitutive activation of MAP kinase and (ii) marked decreases in both IR function and expression which are mediated by PKC-dependent and PKC-independent pathways, respectively.