Statistical analysis of an expanded data base of regions in viral polyproteins and in non-viral proteins that are sensitive to hydrolysis by the protease from human immunodeficiency virus (HIV) type 1 has generated a model which characterizes the substrate specificity of this retroviral enzyme. The model leads to an algorithm for predicting protease-susceptible sites from primary structure. Amino acids in each of the sites from P4 to P4' are tabulated for 40 protein substrates, and the frequency of occurrence for each residue is compared to the natural abundance of that amino acid in a selected data set of globular proteins. The results suggest that the highest stringency for particular amino acid residues is at the P2, P1, and P2' positions of the substrate. The broad specificity of the HIV-1 protease appears to be a consequence of its being able to bind productively substrates in which interactions with only a few Pi or Pi' side-chains need be optimized. The analysis, extended to 22 protein segments cleaved by the HIV-2 protease, delineates marked differences in specificity from that of the HIV-1 enzyme.