The study was designed to investigate the formation of mixed disulfides (PSSG) of protein and glutathione (GSH) in the lens. The possibility that oxidized GSH reacts with lens protein sulfhydryls was examined by incubating tritiated glutathione disulfide ([3H] GSSG) with a solution of dialyzed water-soluble bovine lens crystallins. Gel chromatography on a Sephacryl S-200 column showed that all crystallins have become labeled. The ratios of radioactivity incorporated into each cry stall in relative to the protein concentration (cpm/O.D. ratios) were 0.5, 6, 12 and 11 forα,βH,βL andγ-crystallins, respectively. When [3H] GSSG was covalently linked to and immobilized on derivatized agarose beads (Affi-Gel 10) and then treated with dialyzed lens crystallins part of the radioactivity was released from the gel. Most of the released radioactivity was associated with the protein and was not dialyzable but susceptible to removal by dithiothreitol or glutathione, indicating that it is linked to the protein as PSSG. Dialysis of [3h] PSSG against increasing concentrations of unlabeled GSSG or GSH showed that only GSH could effectively cause the release of radioactivity into the dialysis medium due to the formation of GSSG. It can be concluded that GSSG rather than GSH is involved in the formation of protein-glutathione disulfide and that such a reaction may retard or prevent protein aggregation due to the protein-protein disulfide bond formation that leads to lens opacification.