Previous work (1,2,3) has indicated that the in vivo post-translational modification of the alpha crystallin primary gene product A is due to a specific phosphorylation process involving a serine residue located in a chymotryptic fragment with the sequence ARG-LEU-PRO-SER-ASN-VAL-ASP-GLN-SER-ALA-LEU which corresponds to the residues 119 to 129 of the polypeptide chain. To define which of the two serines is phosphorylated, thepresent experiments were carried out. The32P-labeled chymotryptic fragment was obtained from alpha crystallin isolated from the outer cortex of calf lenses incubated in the presence of [32P]-orthophosphate. By analyses of the products obtained after Edman degradation, utilizing electrophoresis in cellulose TLC plates and radioautography, it was possible to locate the phosphate in the serine residue at position 122 in the polypeptide chain. No phosphate could be detected in the serine residue at position 127.