The cellular transformation induced by viral tyrosine protein kinases may result from the excessive phosphorylation of the normal polypeptide substrates of endogenous cellular tyrosine kinases, from the phosphorylation of proteins that are not normal substrates of cellular tyrosine protein kinases in uninfected cells, or from the phosphorylation of proteins of each type. To differentiate between these possibilities, antibodies to phosphotyrosine were used with immunoblotting to compare the substrates of p60v-src, the transforming tyrosine protein kinase of Rous sarcoma virus (RSV), with those of cellular tyrosine protein kinases. Specifically, the substrates of p60v-src were compared with those of (1) p60c-src, (2) the tyrosine protein kinases activated by the binding of platelet-derived growth factor and (3) normal cellular tyrosine protein kinases in fibroblasts treated with sodium orthovanadate, an inhibitor of phosphatases. Comparison of the patterns observed on the immunoblots with the pattern of phosphotyrosine-containing proteins isolated by immunoaffinity chromatography with antiphosphotyrosine antibodies demonstrated that the proteins detected by Western blotting did indeed contain phosphotyrosine. Cells transformed by a variant of c-src activated by a single point mutation had an almost identical pattern of tyrosine protein phosphorylation as cells transformed by v-src. The several mutations and carboxyl-terminal substitution that differentiate p60v-src from p60c-src appear therefore to affect the enzymatic activity, but not the polypeptide substrate specificity, of the viral protein. In cells transformed by v-src, 27 of the 35 phosphotyrosine-containing proteins were also phosphorylated on tyrosine in normal uninfected fibroblasts treated with sodium orthovanadate. The phosphorylation of the large majority of the substrates of p60v-src can therefore occur in uninfected cells. Nine of the substrates of p60v-src were also phosphorylated by the viral tyrosine protein kinases encoded by the oncogenes, v-abl, v-fps, v-fes, and v-fgr. Together these data are consistent with the idea that viral tyrosine protein kinases induce transformation largely by intervening in cellular regulatory pathways that are normally controlled by tyrosine protein phosphorylation.