The substrate specificity of the human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) proteinases was compared using oligopeptides corresponding to cleavage sites in the Gag and Gag-Pol polyproteins of both viruses. All peptides mimicking cleavage sites at the junction of major functional protein domains were correctly cleaved by both enzymes. However, some other peptides thought to represent secondary cleavage sites remained intact. The kinetic parameters (Km and kcat) obtained for the different substrates showed several hundred-fold variation but were similar for the same substrate.