Vasoactive effects of soluble matrix proteins and integrin-binding peptides on arterioles are mediated by αvβ3 and α5β1 integrins. To examine the underlying mechanisms, we measured L-type Ca2+ channel current in arteriolar smooth muscle cells in response to integrin ligands. Whole-cell, inward Ba2+ currents were inhibited after application of soluble cyclic RGD peptide, vitronectin (VN), fibronectin (FN), either of two anti-β3 integrin antibodies, or monovalent β3 antibody. With VN or β3 antibody coated onto microbeads and presented as an insoluble ligand, current was also inhibited. In contrast, beads coated with FN or α5 antibody produced significant enhancement of current after bead attachment. Soluble α5 antibody had no effect on current but blocked the increase in current evoked by FN-coated beads and enhanced current when applied in combination with an appropriate IgG. The data suggest that αvβ3 and α5β1 integrins are differentially linked through intracellular signaling pathways to the L-type Ca2+ channel and thereby alter control of Ca2+ influx in vascular smooth muscle. This would account for the vasoactive effects of integrin ligands on arterioles and provide a potential mechanism for wound recognition during tissue injury.