The aspartic proteinases are an important family of enzymes associated with several pathological conditions such as hypertension (renin), gastric ulcers (pepsin), neoplastic disease (cathepsins D and E), and AIDS (HIV proteinase). Studies of inhibitor binding are therefore of great importance for design of novel inhibitors for potential therapeutic applications. Numerous X-ray analyses have shown that transition-state isostere inhibitors of aspartic proteinases bind in similar extended conformations in the active-site cleft of the target enzyme. Upon comparison of 21 endothiapepsin inhibitor complexes, the hydrogen bond lengths were found to be shortest where the isostere (P-1-P-1') interacts with the enzyme's catalytic aspartate pair. Hydrogen bonds with good geometry also occur at P-2', and more so at P-3, where a conserved water molecule is involved in the interactions. Weaker interactions also occur at P-2, where the side-chain conformations of the inhibitors appear to be more variable than at the more tightly held positions. At P-2 and, to a lesser extent, P-3, the side-chain conformations depend intriguingly on interactions with spatially adjacent inhibitor side chains, namely P-1' and P-1, respectively. The tight binding at P-1-P-1', P-3, and P-2' is also reflected in the larger number of van der Waals contacts and the large decreases in solvent-accessible area at these positions, as well as their low temperature factors. Our analysis substantiates earlier proposals for the locations of protons in the transition-state complex. Aspartate 32 is probably ionized in the complexes, its charge being stabilized by 1, or sometimes 2, hydrogen bonds from the transition-state analogues at P-1. The detailed comparison also indicates that the P-1 and P-2 residues of substrate in the ES complex may be strained by the extensive binding interactions at P-3, P-1', and P-2' in a manner that would facilitate hydrolysis of the scissile peptide bond.