Site-directed mutagenesis techniques were used to construct defined point mutations within the src gene of the Prague A strain of Rous sarcoma virus. Bisulfite mutagenesis at a Bg/I restriction site in the src gene yielded three mutations which contained the same single base change, a guanine-to-adenine transition. The resulting genomes encoded an src protein containing a substitution of threonine for alanine at amino acid position 433. Transfection of chicken cells with mutagenized DNA did not result in cellular transformation even though the cells produced a pp60src. Immune complexes containing mutant pp60src did not phosphorylate immunoglobulin G heavy chain or casein.