Alterations in activities of one family of proteases, the matrix metalloproteinases (MMPs), have been implicated in primary and metastatic tumor growth, angiogenesis, and pathological degradation of extracellular matrix (ECM) components, such as collagen and laminin. Since hydrolysis of the collagen triple-helix is one of the committed steps in ECM turnover, we envisioned modulation of collagenolytic activity as a strategy for creating selective MMP inhibitors. In the present study, a phosphinate transition state analogue has been incorporated within a triple-helical peptide template. The template sequence was based on the alpha 1(V)436-450 collagen region, which is hydrolyzed at the Gly(439)-Val(440) bond selectively by MMP-2 and MMP-9. The phosphinate acts as a tetrahedral transition state analogue, which mimics the water-bound peptide bond of a protein substrate during hydrolysis. The phosphinate replaced the amide bond between Gly-Val in the P-1-P-1' subsites of the triple-helical peptide. Inhibition studies revealed K-i values in the low nanomolar range for MMP-2 and MMP-9 and low to middle micromolar range for MMP-8 and MMP-13. MMP-1, MMP-3, and MT1-MMP/MMP-14 were not inhibited effectively. Melting of the triple-helix resulted in a decrease in inhibitor affinity for MMP-2. The phosphinate triple-helical transition state analogue has high affinity and selectivity for the gelatinases (MMP-2 and MMP-9) and represents a new class of protease inhibitors that maximizes potential selectivity via interactions with both prime and nonprime active site subsites as well as with secondary binding sites (exosites).