Dynamic remodeling of the actin cytoskeleton plays an essential role in the migration and proliferation of vascular smooth muscle cells. It has been suggested that actin remodeling may also play an important functional role in non-migrating, non-proliferating differentiated vascular smooth muscle (dVSM). In the present study we show that contractile agonists increase the net polymerization of actin in dVSM, as measured by differential ultracentrifugation of vascular smooth muscle tissue and co-staining of single freshly dissociated cells with fluorescent probes specific for G and F actin. Furthermore, induced alterations of the actin polymerization state, as well as actin decoy peptides, inhibit contractility in a stimulus-dependent manner. Latrunculin pretreatment or actin decoy peptides significantly inhibit contractility induced by a phorbol ester or an alpha agonist, but these procedures have no effect on contractions induced by KCl. Aorta dVSM expresses alpha smooth muscle actin, beta actin, nonmuscle gamma actin and smooth muscle gamma actin. Incorporation of isoform-specific cell-permeant synthetic actin decoy peptides, as well as isoform-specific probing of cell fractions and 2-D gels, demonstrate that actin remodeling during alpha agonist contractions involves remodeling of primarily gamma actin and, to a lesser extent, beta actin. Taken together, these results show that net isoform-dependent and agonist-dependent increases in actin polymerization regulate vascular contractility.