Polyomavirus middle T antigen mediates transformation of cells, at least in part, by its association with and activation of the intrinsic protein tyrosine kinase activity of pp60c-src. pp60c-src, by analogy with pp60v-src, elicits cell proliferation through a signal transduction pathway that includes p21c-ras. Therefore, we tested the possibility that middle T antigen acts upstream of and in the same proliferative signaling pathway as p21c-ras. Co-transfection of Rat-2 cells with plasmids expressing human Krev-1, a dominant suppressor of Ki-ras transformation, and mT antigen resulted in a dose-dependent reduction of mT antigen-induced foci. Krev-1 did not affect the transforming activity of SV40 large T antigen, demonstrating that the transformation-suppressing activity of Krev-1 is specific. To determine the effect of Krev-1 on stably transformed cell lines, Krev-1 DNA was introduced into middle T antigen-transformed Rat-2 cells along with a G418 resistance marker. Of the G418-resistant colonies examined, 1% were morphologically untransformed. Characterization of several morphological revertants revealed that, with the exception of one cell line, all of the cell lines expressed middle T antigen, which was associated with pp60c-src, whose tyrosine kinase activity was similar to that found in the parental transformed cell lines. To determine whether other phenotypic traits associated with transformation were altered in these cell lines, their growth rates and ability to form colonies in agar suspension were examined. The majority of the revertants had longer doubling times, and grew less efficiently in agar suspension compared with their transformed parents. A direct correlation was observed between Krev-1 RNA and protein expression and the efficiency with which the revertants formed colonies in suspension. These results suggest that p21c-ras lies downstream of middle T antigen and pp60c-src in the same proliferative signal transduction pathway.