The differences in substrate specificity between Moloney murine leukemia virus protease (MuLV PR) and human immunodeficiency virus (HIV) PR were investigated by site-directed mutagenesis. Various amino acids, which are predicted to form the substrate binding site of MuLV PR, were replaced by the equivalent ones in HIV-1 and HIV-2 PRs. The expressed mutants were assayed with the substrate Val-Ser-Gln-Asn-Tyr decreases Pro-Ile-Val-Gln-NH2 (decreases indicates the cleavage site) and a series of analogs containing single amino acid substitutions in positions P4(Ser) to P3'(Val). Mutations at the predicted S2/S2' subsites of MuLV PR have a strong influence on the substrate specificity of this enzyme, as observed with mutants H37D, V39I, V54I, A57I, and L92I. On the other hand, substitutions at the flap region of MuLV PR often rendered enzymes with low activity (e.g. W53I/Q55G). Three amino acids (His-37, Val-39, and Ala-57) were identified as the major determinants of the differences in substrate specificity between MuLV and HIV PRs.