A series of mutations, first identified in protease inhibitor-resistant HIV-1 viral isolates, were introduced into HIV-1 PR as individual substitutions. Mutants containing R8K, V32I, V82T, I84V, G48V/L90M, or V82T/I84V substitutions were analyzed for differences in substrate preference and catalytic efficiency using a set of single amino acid substituted HIV-1 CA-NCa cleavage site peptides. All mutants exhibited wild-type preference for large hydrophobic residues, especially Phe, in the P1' substrate position. Only the R8K and V32I mutants showed significant differences in subsite selection compared to wild-type enzyme. In a parallel study, the individual mutations R10K, L12V, I44V, A60M, I71V, and I108V were introduced into RSV PR. These amino acid positions are structurally equivalent to Arg8, Leu10, Val32, Met46, Ile54, and Ile84 in HIV-1 PR, respectively, which mutate in drug-resistance. The RSV R10K substitution significantly altered substrate specificity and catalytic rate, compared to wild-type, in a manner similar to that of the HIV-1 R8K mutant. Crystal structures of the RSV PR R10K, I44V, I71V, and Il08V mutant enzymes presented here indicate that each of these substitutions has little effect on the overall structure of the respective enzymes. Taken together, these data provide an explanation for the reported in vivo predilection for selection of large hydrophobic residues in the P1' substrate position of second locus mutations in the Gag polyprotein PR cleavage sites. The data also suggest that the selection of resistant enzymes is not simply limited to loss of binding to inhibitor but affects other steps in proteolysis.