GTPase-activating protein interactions with the viral and cellular Src kinases. GTPase-activating protein (GAP), which regulates the activities of Ras proteins, is implicated in mitogenic signal transduction by growth-factor receptors and oncoproteins with tyrosine kinase activity. Oncogenic viral Src (p60v-src) encoded in Rous sarcoma virus possesses elevated tyrosine kinase activity compared with its nononcogenic normal homolog, cellular Src (p60c-src). To examine molecular interactions between GAP and the two Src kinases, immunoprecipitates of Src or GAP prepared from cell lystates were resolved by gel electrophoresis and analyzed by an immunoblot procedure with antibodies to GAP or Src used as probes. Results suggest that p60c-src is associated with a complex containing GAP in immunoprecipitates from lysates of normal rat and chicken cells. However, GAP is not phosphorylated in p60c-src immunoprecipitates subjected to in vitro kinase reactions. By contrast, GAP undergoes tyrosyl phosphorylation in vitro when immunoprecipitates of p60v-src prepared from transformed cell lysates are incubated with ATP. Our findings suggest that p60v-src and p60c-src associate with complexes containing GAP and provide a biochemical link between both kinases and GAP/Ras signal transduction pathways. These results are consistent with the hypothesis that GAP has a role in mediating normal functions of p60c-src as well as oncogenic activities of p60v-src. 