The avian sarcoma virus (ASV) protein responsible for cellular transformation in vitro and sarcomagenesis in animals was studied structurally with special reference to the sites of phosphorylation on the polypeptide. The product of the ASV src gene, pp60src, is a phosphoprotein of 60,000 daltons. We found that pp60src contained two major sites of phosphorylation, one involving phosphoserine and the other involving phosphothreonine and possible addtional minor sites of phosphorylation. By using N-formyl[35S]methionyl-tRNAf as a radiolabeled precursor in the cell-free synthesis of the src protein in conjunction with partial proteolysis mapping, we determined that the major phosphoserine residue was located on the amino-terminal two-thirds of the molecule and that the phosphothreonine was located on the carboxy-terminal third. We further determined that the phosphorylation of pp60src in cell extracts involved at least two protein kinases, the one that phosphorylated the major serine site being cyclic AMP dependent and the other, acting on the threonine residue, being a cyclic nucleotide-independnet phosphotransferase. Finally, analysis of the pp60src isolated from cells infected with a temperature-sensitive src gene mutant of ASV revealed that phosphorylation of the major threonine residue was severely reduced when infected cells were grown at the nonpermissive temperature, whereas a phosphorylation pattern characteristic of the wild-type pp60src was observed at the permissive temperature. As pp60src has an associated protein kinase activity, the possible involvement of phosphorylation-dephosphorylation reactions in the functional regulation of ASV transforming protein enzymatic activity is discussed.