To understand the differences in the binding specificities within the aspartic proteinase family of enzymes, we have carried out studies to determine the inhibition constants of a set of related compounds with various members of the human enzyme family. The inhibition constants (Ki values) were determined by competitive inhibition of the hydrolysis of chromogenic octapeptide substrates in the pH range of 3-5. For comparison, inhibition of monkey renin was studied by RIA at pH 6.0. All inhibitors were based on the general structure 4-(morpholinylsulfonyl)-L-Phe-P2-(cyclohexyl)Ala psi[isostere]-P1'-P2'. The isosteric replacements of the scissile peptide bond included difluorohydroxyethylene, 1,2-diols, 1,3-diols, and difluoroketones. Side chain substituents in P2 include hydrogen, allyl, ethylthio, (methoxycarbonyl)methyl, N-methylthiouridobutyl, imidazolylmethyl, and 4-amino-2-thiazolylmethyl. Our measurements have identified potent and selective inhibitors which are useful in evaluating the differences in the specificities among selected enzymes of this family.