The p2 gag peptide (AEAMSQVTNTATIM) processed from HIV-1 Pr55 gag by HIV-1 protease was identified as a suicide inhibitor of the enzyme (Ki = 30 μM and IC 50 = 10 μM for the synthetic peptide substrate, succinyl-SQNYPIVQ), and potently inhibited the proteolytic cleavage of the viral precursor protein (Pr55 gag ) into functional structural units (p17 gag and p24 gag )in vitro.The nonapeptide (AEAMSQVTN) derived fromN-terminus of the p2 gag peptide exhibits a potent inhibitory action on HIV-1 protease, but the other peptides (AEAMSQ, AEAMSQV, AEAMSQVT, VTN and VTNTATIM) do not. It was determined by exclusion gel chromatography that HIV-1 protease after treatment of the synthetic p2 gag peptide dissociated from the active dimeric form to an inactive monomeric form. The p2 gag peptide and HIV-1 protease were also detected in HIV-1 viral particles using both matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI TOF-MS) and western immunoblot analyses. Taken together, these results suggest that the p2 gag peptide is the inhibitor of preventing dimerization of HIV-1 protease and that the enzyme activity is completely suicide inhibited with the accumulated p2 gag peptide producing by the processing of Pr55 gag during viral maturation.