The cAMP-dependent phosphorylation of α-crystallin was investigated. The major products of in vitro phosphorylation of total bovine lens homogenate are the αA1 and αB1 polypeptides, but in addition a minor labeled spot is present which might correspond with a double phosphorylated αB chain. It is demonstrated that the A1 and B1 subunits of α-crystallin from bovine eye lenses are solely the result of phosphorylation of the primary gene products αA2 and αB2, respectively, as judged from the stoichiometry of the phosphate content of these polypeptides. Both the in vitro and in vivo phosphorylation sites of the A chain of bovine α-crystallin were determined and found to be the same. After in vitro incubation the majority of the 32P label was found in the tryptic peptides T17a and T16-T17a, the latter being the result of incomplete tryptic cleavage between T16 and T17a. The in vivo phosphorylation site is also located in T17a, as could be concluded from the retention times on reversed-phase HPLC of T16–17a and T17a from αA1 as compared to those from αA2, and from the differences in their mobilities on high-voltage paper electrophoresis at pH 6.5. Furthermore, both T17a and T16–17a of αA1 contain approximately 1 mol phosphate/mol peptide. Thermolytic digestion of T16–17a of both αA2 and αA1, followed by separation on RP-HPLC, demonstrated that Ser-122 is the phosphorylation site of the A chain of bovine lens α-crystallin. The replacement of this phosphorylation site or the lack of basic amino acids at the N-terminal side of Ser-122 in some vertebrate species apparently results in the absence of phosphorylation of α-crystallin A both in vitro and in vivo.