Inhibitors of human immunodeficiency virus-1(HIV-1) proteinase have been used for several years to treat acquired immunodeficiency syndrome patients. Despite intensive research, however, the substrate specificity of this enzyme is not completely elucidated. Here, we assessed the HIV-1 proteinase P(4) to P(2) substrate specificity using a bacterial screening system. In this system, the bacterial enzyme beta-galactosidase has been transformed into an HIV-1 proteinase substrate by insertion of the p6/PR cleavage site. Consequently, HIV-1 processing can be determined by measuring the beta-galactosidase activity on X-gal plates and by examination of the extent of cleavage of the beta-galactosidase protein itself. We screened a library containing randomized sequences at the P(4) to P(2) positions and found strong preferences for Thr, Ser, and Pro at P(4), for Leu, Met, and Phe at P(3), and for Ser, Met, and Leu at P(2). The frequent observations of Thr at P(4) and Ser at P(2) extend previous findings and offer the possibility of producing inhibitors with different properties. These new data on HIV proteinase specificity illustrate the usefulness of random libraries in the genetic screening system. This approach can be applied to examine any proteinase that has a recognition site extending across several amino acids.