Macrocyclic inhibitor 1 {methyl[cyclo-7[(2R)-((N-valyl)amino)-2-(hydroxyl-(1S)-1-methyoxycarbonyl-2-phenylethoxy)phosphinyloxyethyl]-1-naphthaleneacetamide]sodium salt} was designed according to the conformation of the acyclic analogue Iva-L-Val-L-Val-L-Leu(P)-(O)Phe-OMe [Leu(P) = the phosphinic acid and analogue of L-leucine; (O)Phe = L-3-phenyllactic acid; OMe = methyl ester] (4) bound to penicillopepsin, by linking the P1 and P3 side chains of the penicillopepsin inhibitor. This compound and its two acyclic derivatives, {methyl(2S)-[1-(((N-Formyl)-L-valyl)amino-2-(2-naphthyl)ethyl)hydroxyphosphinyloxy]-3-phenylpropanoate, sodium salt} (2) and {methyl(2S)-[1-(((N-(1-naphthaleneacetyl))-L-valyl)aminomethyl)hydroxyphosphinyloxy]-3-phenylpropanoate, sodium salt} (3), have been synthesized and evaluated as inhibitors of penicillopepsin. Their binding affinity to the enzyme was found to be inversely related to the predicted degree of conformational flexibility across the series : 3 (K-i = 110 mu M), 2 (K-i = 7.6 mu M), 1 (K-i = 0.8 mu M). The X-ray crystallographic structures of penicillopepsin complexed with the macrocyclic peptidyl phosphonate 1 and with its two derivatives 2 and 3 have been determined and refined to crystallographic agreement factors R (=Sigma\\F-o\ - \F-c\\/Sigma\F-o\) of 15.9%, 16.0%, and 15.2% and R-free of 19.8%, 20.3%, and 21.4%, respectively. The intensity data for all complexes were collected to 1.5 Angstrom resolution. One 1.25 Angstrom resolution data set was obtained for the complex with 1 at 110 K; the structure was refined to an R factor of 15.0% (R-free of 19.7%) The binding interactions that 1 and 2 make with penicillopepsin are similar to those that have been observed for other transition-state mimics with aspartyl proteinases. On the other hand, the acyclic Linear inhibitor 3 exhibits distinctive binding to penicillopepsin with the phosphonate group shifted similar to 3.0 Angstrom from the average position observed for the other complexes. These structural results show that the macrocyclic constraint of 1 enhances its binding affinity over those of the acyclic analogues but the differences in the observed bound dispositions mean that the effect has yet to be quantified.