The structure of the catalytic domain of human memapsin 2 bound to an inhibitor OM00-3 (Glu-Leu-Asp-Leu*Ala-Val-Glu-Phe, K_i = 0.3 nM, the asterisk denotes the hydroxyethylene transition-state isostere) has been determined at 2.1 Å resolution. Uniquely defined in the structure are the locations of S₃‘ and S₄‘ subsites, which were not identified in the previous structure of memapsin 2 in complex with the inhibitor OM99-2 (Glu-Val-Asn-Leu*Ala-Ala-Glu-Phe, K_i = 1 nM). Different binding modes for the P₂ and P₄ side chains are also observed. These new structural elements are useful for the design of new inhibitors. The structural and kinetic data indicate that the replacement of the P₂‘ alanine in OM99-2 with a valine in OM00-3 stabilizes the binding of P₃‘ and P₄‘.