Two techniques were used to search for the polypeptide encoded by the avian sarcoma virus (ASV) src gene. First, antiserum from rabbits bearing ASV-induced fibrosarcomas was used to immunoprecipitate a transformation-specific antigen from ASV-transformed chick embryo fibroblasts. This antigen has an apparent molecular weight (Mr) of 60,000. Second, the 3' one-third of the ASV genome, selected by oligo(dT)-cellulose chromatography and sucrose gradient sedimentation, was translated in a mRNA-dependent reticulocyte cell-free lysate. This RNA species programmed the synthesis of a polypeptide that comigrated with the transformation-specific antigen of Mr 60,000 immunoprecipitated from transformed cells. The methionine-containing tryptic peptides from the polypeptides of Mr 60,000 obtained from translation in vitro and from immunoprecipitation were found to be identical upon two-dimensional fractionation.