The radiolytic decomposition products of [35S]-methionine have been used to radiolabel proteins in vitro. The process occurs in a time-, temperature-, and pH-dependent manner. Maximum labeling of bovine serum albumin occurs after a 24 h incubation at 37°C and pH 8.5. Once incorporated, the radiolabel cannot be removed by extended incubation at various temperatures, multiple freeze/thaw cycles, or boiling, indicating that the 35S moiety is covalently attached to the protein. A wide variety of proteins have been radiolabeled. The method is simple to perform and yields radiolabeled proteins of high specific activity. © 1992.