A series of 47 N-truncated reduced bond inhibitors, systematically modified at individual positions (P1, P'1, P'2, P'3, and P'4), were synthesized and used to map the subsite preferences of HIV-1 protease. The tight binding inhibitor of HIV-1 protease t-butoxycarbonyl-Phe-[CH2NH]Phe-Glu-Phe-NH2 (Ki = 0.2 nM) was chosen as the parent structure for further modifications. The P'2 glutamic acid was found to fit well into the S'2 subsite of the protease. The conformational restriction of any phenylalanine residue or saturation of more than one phenylalanine side chain in P'1 or P'3 lead is to a large Ki increase. Introduction of tyrosine in the P1 position improves the binding by an order of magnitude. The S'4 subsite of the protease was shown to accommodate large structural changes in the inhibitor at this position. Therefore P'4 may serve as an ideal region for further modification in order to improve bioavailability of this type of compound. An improved method of direct comparison of tight binding inhibitors with subnanomolar Ki values has been described.