To test whether an observed age-related increase in the level of oxidized protein in rat liver is due to a decrease in the activity of the multicatalytic proteinase (MCP), this protease was isolated from liver of young (8-month-old) and old (24-month-old) male Fischer 344 rats. Three peptidase activities of the MCP were assayed using fluorogenic peptides: trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide hydrolase. Only peptidylglutamyl-peptide hydrolase activity declined with age, with protease from old animals exhibiting approximately 50% of the activity of that from young animals. Bidimensional gel electrophoresis and thermostability studies did not reveal age-related structural modifications of the MCP subunits. Peptidylglutamyl-peptide hydrolase activity and trypsin-like activity were sensitive to metal-catalyzed oxidation. In some preparations, a 95-kDa protein that has been identified as the heat shock protein 90 copurified with the MCP. In the presence of HSP 90, trypsin-like activity is protected from oxidative inactivation and chymotrypsin-like activity is slightly activated. Peptidylglutamyl-peptide hydrolase activity remained sensitive to oxidation in protease isolated from young rats, but that from old rats was resistant to oxidative inactivation. Furthermore, addition of rat HSP 90 to rat liver MCP (purified from 8-month-old animals and free of contaminating HSP 90) was found to protect trypsin-like activity from oxidative inactivation.