A pathway for the regulation of vascular tone appears to involve coupling between integrins and extracellular matrix proteins or their fragments and the subsequent modulation of ion movement across the smooth muscle cell membrane. Here, we report that the activation of L-type voltage-activated Ca2+channels occurs through a novel interaction of &agr;4&bgr;1integrin with peptides containing the Leu-Asp-Val (LDV) integrin–binding sequence, which is found in the CS-1 region of an alternately spliced fibronectin variant. Experiments were conducted on arterioles isolated from rat skeletal muscle. Arterioles exhibited sustained concentration-dependent vasoconstriction to LDV peptides but not to Leu-Glu-Val (LEV) control peptides. The constriction was associated with increased smooth muscle cell [Ca2+]i, as measured by using fura 2. The response could be inhibited with a function-blocking anti–&agr;4integrin antibody. Removal of the endothelium did not alter the vasoconstrictor response. Further experiments demonstrated that the vasoconstriction was abolished by the L-type Ca2+channel inhibitor nifedipine and the Src family kinase inhibitor PP2. In studies of isolated smooth muscle cells using whole-cell patch-clamp methods, the L-type current was enhanced by the LDV but not LEV peptide and was blocked by PP2 or antibodies to &agr;4integrin. Collectively, these data indicate that activation of &agr;4&bgr;1integrin leads to enhanced influx of Ca2+through L-type channels by activating a tyrosine kinase pathway, leading to vasoconstriction. Involvement of integrins in the modulation of vascular tone may be particularly important in vascular responses to mechanical signals, such as pressure and flow, and to tissue injury after damage to the extracellular matrix.