The full cycle of design, synthesis, and enzymatic and crystallographic evaluation of a structure-based inhibitor of thermolysin is described. Using the structure of the complex of thermolysin with Cbz-Gly(P)-Leu-Leu (K-i = 9 nM; "Gly(P)" = NHCH2PO2-) as the starting point, we designed the macrocyclic phosphonamidate (S,S)-1 as a conformationally constrained derivative. The chroman linking unit was designed to rigidify the peptide analog while avoiding unfavorable steric interactions with the enzyme. (S,S)-1 was synthesized by a route that provided all of the stereoisomers in pure form; the acyclic control compounds 2 and 3 were also prepared. The binding affinity of (S,S)-1 (K-i = 4 nM) is enhanced by 2.3 kcal/mol in comparison to 3 (K-i = 190 nM); the other diastereomers of 1 are considerably less potent (K-i greater than or equal to 500 nM). Crystallographic analysis of the complexes between thermolysin and (S,S)-1 and (S)-2 demonstrated that the anticipated mode of binding of (S,S)-1 was fundamentally correct, while revealing some discrepancies with the original model. The structural studies also showed that the chroman moiety in the acyclic analog (S)-2 binds in a different orientation than in the macrocycle, an observation that is important for interpreting the differences in affinity between the macrocyclic inhibitor and the control compounds.