Integrin alphavbeta3 and matrix metalloprotease-2 (MMP-2) are two established molecular targets of angiogenesis. Basic understanding of various forms of functional interaction of integrin alphavbeta3 and active MMP-2 may be used to develop therapeutic approaches. Based upon the idea that integrins are present on the surface of invasive cells and MMP-2 may be localized to this and other cell-surface receptors, we investigated the hypothesis that integrin binding will alter cleavage of MMP-2 substrates. To investigate this hypothesis, integrin-binding and MMP-2 cleavable motifs were combined in a single peptide, MMP-RGD, designed with fluorescent probes for monitoring peptide cleavage. MMP-RGD was bound to integrin alphavbeta3 with equal affinity compared to the integrin-binding motif and was cleaved with equal specificity by active MMP-2. MMP-RGD bound to human umbilical vein endothelial cells (HUVECs). MMP-2 from HUVECs cleaved MMP-RGD, but the cleavage was not altered due to integrin binding. Our results indicate that integrin alphavbeta3 and active MMP-2 may not be as functionally collaborative for substrate cleavage as expected based on the current knowledge of their cell surface colocalization.