Pepsin C, one of the minor gastric proteases of the pig, is fairly similar in most of its properties to the major enzyme, pepsin. The most marked difference between the two enzymes is the inability of pepsin C to catalyse the hydrolysis of acetyl-phenylalanyl-diiodotyrosine, a good synthetic substrate for pepsin. It was of interest to try to find which group(s) in pepsin C was involved in the catalytic activity of the enzyme and to make a comparison between this and the active site region of pepsin itself. The active site of pepsin C was investigated by the technique of 'affinity labelling'. Diazoacetyl norleucine methyl ester was found to inactivate the enzyme very rapidly and irreversibly in the presence of cupric ions at pH values above 4.5. The inactivation was specific, in that one mole of inhibitor was incorporated per mole of enzyme when the enzyme had lost 100% of its activity. Evidence was found for the existence of a binding site for the inhibitor and it was found that a competitive inhibitor protected the enzyme against inactivation by the diazo compound. The irreversible inhibitor did not interact to any great extent with denatured pepsin C, all of which suggests that diazoacetyl norleucine methyl ester inactivates the enzyme by reaction at the active site.