Oligopeptides that correspond to proteolytic cleavage site junctions of the native Gag and Pol proteins are specifically cleaved by retroviral aspartate proteases (PRs). The role of the flap subdomain of the PR of the human spumaretrovirus (HSRV) and of substrate peptides in cleavage site specificity was analyzed by site-directed mutagenesis. Native and mutant peptides were subjected to proteolysis by the authentic and mutated recombinant viral enzyme. The results reveal that Glu residue 54 of the HSRV PR is an essential specificity determinant for proteolytic processing of the structural proteins. Peptides that represent in vivo cleavage sites were susceptible to proteolysis by the recombinant HSRV PR, but one peptide located at the junction between the PR and reverse transcriptase domains was completely resistant to cleavage. Thus the data indicate that a proteolytic cleavage between these domains does not occur in vivo. Naturally occurring and mutant forms of the cleavage-resistant peptide were therefore analyzed by circular dichroism to determine if differences existed in the secondary structures of the peptides that did or did not serve as substrates. The data show that differences in the secondary structure of the native and mutant peptides analyzed does not seem to play a crucial role for cleavage site specificity in HSRV PR. Instead highly conserved hydrophobic residues at distinct positions of the HSRV cleavage site junctions contribute to the specificity observed as reported for HIV-1 PR.