An aminopeptidase preferentially hydrolyzing Leu- or Ala-Gly-Gly was purified from rat brain cytosol and detailed studies have been performed on its substrate specificity and the effects of inhibitors. The enzyme was devoid of di- and oligopeptidase contamination. Biologically active tripeptides such as Met-Leu-Tyr (chemotactic factor), Gly-His-Lys (liver growth factor) and Thr-Val-leu central nervous system tripeptide) were hydrolyzed at rates 0.05-0.15-times that of Leu-Gly-Gly. Melanostatin (Pro-Leu-GlyNH2) did not serve as a substrate. Substrates bearing N-terminal charged groups, or ones with proline in positions 2 or 3, or those with D-amino acid in positions 1 or 2, or with C-terminal CONH2, were poorly hydrolyzed or did not act as substrates, providing information on subsites involved in enzyme catalysis. The enzyme was inhibited competitively by bestatin (Ki 10-7 M) and by Captopril (2.5.10-7 M, D-3-thio-2-methylpropanyl proline) and by low concentrations of Zn2+ or PCMB, and at higher concentrations by TPCK and PMSF. Inhibition was observed for the chemotactic factor (I50 13 microM) and for the central nervous system tripeptide (195 microM). The enhanced action of Captopril was attributed to the presence of the -SH and -CH3 groups, since inhibition was shared by di- and tripeptides with proline in positions 2 and 3. The specificity pattern of brain enzyme was different from that reported for kidney and intestine.