The amino acid residues important for the catalytic activity of the Cl- -activated arginine aminopeptidases from human erythrocytes and rat liver were studied using enzyme modification. The general inhibition characteristics were similar with both enzymes. Inactivation with 5,5'-dithiobis-(2-nitrobenzoic acid) revealed one essential SH-group per active enzyme unit in both aminopeptidases. L-Arginyl-L-phenylalanine and N-L-arginyl-2-naphthylamide protected the enzymes against inactivation by DTNB, the former substrate being more effective. The rat liver enzyme was more sensitive to DTNB than the erythrocyte enzyme. Titration with DTNB revealed only fast reacting SH-groups in rat liver APB (mean 7.8). The erythrocyte enzyme, however, revealed SH-groups which reacted fast with low concentrations of DTNB, while high concentrations of DTNB or SDS treatment were needed to reveal all enzyme SH-groups (mean 8.0). The presence of at least one essential imidazole group in the erythrocyte enzyme was indicated by photooxidation in the presence of methylene blue, as previously found with the rat liver enzyme (Mäkinen and Hopsu-Havu, 1967, Enzymologia 32, 333). The pH dependence curves of both enzymes also supported the presence of SH- and imidazole groups at or near the active site. Thus, the functional groups identified were the same for both enzymes. Neither enzyme had essential COOH or arginyl groups and they did not contain any zinc. The absence of Zn suggests that the reaction mechanism recently presented by other authors, based on the presence of Zn in the active center, does not apply to the Cl- -activated arginine aminopeptidases. Accordingly, this enzyme group cannot be classified to metallopeptidases.