An important role for the enzyme ADP-ribosylcyclase (ADPR cyclase) and its downstream targets, the ryanodine receptors (RyR), is emerging for a variety of vascular systems. We hypothesized that the ADPR cyclase/RyR pathway contributes to regulation of renal vasomotor tone in vivo. To test this, we continuously measured renal blood flow (RBF) in anesthetized Sprague-Dawley rats. Infusion of the ADPR cyclase inhibitor nicotinamide intrarenally at low doses inhibits angiotensin II (Ang II)- and norepinephrine (NE)-induced vasoconstriction by 72% and 67% (P<0.001). RBF studies in rats were extended to mice lacking the predominant form of ADPR cyclase (CD38). Acute renal vasoconstrictor responses to Ang II and NE are impaired by 59% and 52%, respectively in anesthetized CD38-/- mice compared to wild type controls (P<0.05). Intrarenal injection of the RyR activator FK506 decreases RBF by 22% (P<0.03). Furthermore, RyR inhibition with ruthenium red attenuates Ang II and NE responses by 50% and 59%, respectively (P0.01). Given at higher doses, nicotinamide increases basal RBF by 22% (P<0.001). Non-receptor-mediated renal vasoconstriction by L-type voltage-gated Ca²⁺ channels is also dependent on ADPR cyclase and RyRs. Nicotinamide and ruthenium red inhibit constriction by the L-type channel agonist Bay-K8644 by 59% (P<0.02) and 63% (P<0.001). We conclude that: 1) ADPR cyclase activity contributes to regulation of renal vasomotor tone under resting conditions, 2) renal vasoconstriction induced by G-protein coupled receptor agonists Ang II and NE is mediated in part by ADPR cyclase and RyRs, and 3) ADPR cyclase and RyRs participate in L-type channel-mediated renal vasoconstriction in vivo.