Thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) are potent vasoconstrictors whose contractile effects are mediated by increases in cellular calcium. Stable analogues of these compounds have shown calcium ionophore activity at high concentrations. To determine if effects of TXA2/PGH2 analogues of 42Ca2+ fluxes are receptor mediated, the effects of the stable TXA2/PGH2 mimetic U46619 and the TXA2/PGH2 receptor antagonist I-PTA-OH on 45Ca2+ fluxes in cultured human vascular smooth muscle cells were studied. The smooth muscle cells were cultured from human saphenous vein explants, and they retained the morphologic and immunologic characteristics of vascular smooth muscle cells. U46619 stimulated 45Ca2+ efflux in a dose-dependent manner with an EC50 of 398 ± 26 nM (n = 4). The maximal 45Ca2+ efflux in response to U46619 (5 μM) was significantly greater (p = 0.006) than the 45Ca2+ efflux induced by KCl (40 mM). I-PTA-OH inhibited the U46619-induced 45Ca2+ efflux but had no effect on KCl-induced 45Ca2+ efflux. These results suggest that the effects of U46619 in increasing vascular smooth muscle cell calcium efflux are receptor mediated. Furthermore, vascular smooth muscle cells with functional TXA2/PGH2 receptors were cultured from human saphenous veins and provide a potentially useful in vitro system for the further study of TXA2/PGH2 receptor-mediated phenomena in human vascular tissue.