Previous studies identified the amino acid changes involved in the activation of p60c-src and revealed that the activation accompanies an alteration of its tyrosine-phosphorylation site. We show here that p60c-src that had been converted to transforming protein by amino acid substitution of the c-src gene either at position 63, 95 and 96, or 338 (J. Kato, T. Takeya, C. Grandori, H. Iba, J. B. Levy, and H. Hanafusa, Mol. Cell. Biol. 6:4155-4160, 1986) and encoded in a Rous sarcoma virus variant was phosphorylated on both Tyr-416 and Tyr-527 in chicken embryo fibroblasts. The results obtained from protease V8 analysis, tryptic peptide mapping, and fractionation with nonionic detergent indicated that the p60 of each variant was present in two forms in the population of the virus-infected cells; one was phosphorylated on Tyr-416, and the other was phosphorylated on Tyr-527. On the other hand, colonies isolated in soft agar contained exclusively p60 of which only Tyr-416 was phosphorylated. These results implied that the limited population of p60 was activated in these Rous sarcoma virus variant-infected chicken embryo fibroblasts and that the activated p60 was concentrated in transformed cells. Furthermore, these two forms of p60 differed in their affinity for the detergent-insoluble cellular matrix in spite of their identical primary amino acid sequences, suggesting that the effect of alteration of the tyrosine phosphorylation site was coupled with the degree of stability of this association.