MMP-2 (matrix metalloproteinase 2) contains a CBD (collagen-binding domain), which is essential for positioning gelatin substrate molecules relative to the catalytic site for cleavage. Deletion of the CBD or disruption of CBD-mediated gelatin binding inhibits gelatinolysis by MMP-2. To identify CBD-binding sites on type I collagen and collagen peptides with the capacity to compete CBD binding of gelatin and thereby inhibit gelatinolysis by MMP-2, we screened a one-bead one-peptide combinatorial peptide library with recombinant CBD as bait. Analyses of sequences from the CBD-binding peptides pointed to residues 715-721 in human alpha1(I) collagen chain as a binding site for CBD. A peptide (P713) including this collagen segment was synthesized for analyses. In SPR (surface plasmon resonance) assays, the CBD and MMP-2(E404A), a catalytically inactive MMP-2 mutant, both bound immobilized P713 in a concentration-dependent manner, but not a scrambled control peptide. Furthermore, P713 competed gelatin binding by the CBD and MMP-2(E404A). In control assays, neither of the non-collagen binding alkylated CBD or MMP-2 with deletion of CBD (MMP-2DeltaCBD) bound P713. Consistent with the exodomain functions of the CBD, P713 inhibited approximately 90% of the MMP-2 gelatin cleavage, but less than 20% of the MMP-2 activity on a peptide substrate (NFF-1) which does not require the CBD for cleavage. Confirming the specificity of the inhibition, P713 did not alter MMP-2DeltaCBD or MMP-8 activities. These experiments identified a CBD-binding site on type I collagen and demonstrated that a corresponding synthetic peptide can inhibit hydrolysis of type I and IV collagens by competing CBD-mediated gelatin binding to MMP-2.