Renal hemodynamic studies tested the hypothesis that CD38 and superoxide anion (O₂^.-) participate in vasoconstriction produced by activation of thromboxane prostanoid (TP) receptors in the mouse kidney. CD38 is the major mammalian ADP-ribosyl cyclase contributing to vasomotor tone through generation of cyclic ADP-ribose, a second messenger that activates ryanodine receptors to release Ca2+ from the sarcoplasmic reticulum in vascular smooth muscle cells. We evaluated whether the stable thromboxane mimetic U-46619 causes less pronounced renal vasoconstriction in CD38 deficient mice and the involvement of O₂^.- in U-46619-induced renal vasoconstriction. Our results indicate that U-46619 activation of TP receptors causes renal vasoconstriction in part by activating cADP-ribose signaling in renal resistance arterioles. Based on maximal renal blood flow and renal vascular resistance responses to bolus injections of U-46619, CD38 contributes 30-40% of the TP receptor-induced vasoconstriction. We also found that the anti-oxidant superoxide dismutase mimetic tempol attenuated the magnitude of vasoconstriction by U-46619 in both groups of mice, suggesting mediation by O₂^.-. The degree of tempol blockage of U-46619-induced renal vasoconstriction was greater in wild-type mice, attenuating renal vasoconstriction by 40% as compared to 30% in CD38 null mice. In other studies U-46619 rapidly stimulated O₂^.- production (DHE fluorescence) in isolated mouse afferent arterioles, an effect abolished by tempol. These observations provide the first in vivo demonstration of CD38 and O₂^.- involvement in the vasoconstrictor effects of TP receptor activation in the kidney and in vitro evidence for TP receptor stimulation of O₂^.- production by the afferent arteriole.