Pyroglutamyl aminopeptidase I (PAP-I) is known for specifically removing the L-pyroglutamate (L-pGlu) residue from the amino terminus of L-pGlu proteins and peptides. In general, substrate recognition of PAP-I as to L-pGlu moiety is tightly regulated. However, we recently identified PAP-I as a metabolic enzyme of an organic nitrate compound, RS-7897, which contains L-2-oxothiazolidine-4-carboxylic acid (L-OTCA). L-OTCA is a latent sulfhydryl group, which has moiety structurally related to L-pGlu. In this study, we investigated the substrate specificity of PAP-I toward modified L-pGlu-containing substrates using recombinant rat, mouse and human PAP-Is. PAP-I was tolerant of replacement of a carbon atom at the 4-position of the L-pGlu moiety by a sulfur atom (L-OTCA), an oxygen atom (L-2-oxooxazolidine-4-carboxylic acid, L-OOCA) and an NH group (L-2-oxoimidazolidine-4-carboxylic acid, L-OICA). The K(m) values for rat PAP-I in hydrolyzing L-pGlu-L-Ala, L-OTCA-L-Ala, L-OOCA-L-Ala and L-OICA-L-Ala were 0.057, 0.43, 0.71 and 0.42 mM, respectively. Similar results were observed in mouse and human PAP-Is as well. Moreover, the hydrolysis of RS-7897 in rat and mouse liver cytosols were both completely inhibited by an antibody against rat PAP-I, strongly suggesting that PAP-I is solely involved in the hydrolysis of L-OTCA-containing compounds in rat and mouse liver cytosols.