Physiological concentrations of [Arg⁸]vasopressin (AVP; 10-500 pM) stimulate oscillations of cytosolic free Ca²⁺ concentration (Ca²⁺ spikes) in A7r5 vascular smooth muscle cells. We previously reported that this effect of AVP was blocked by a putative phospholipase A₂ (PLA₂) inhibitor, ONO-RS-082 (5 µM). In the present study, the products of PLA₂, arachidonic acid (AA), and lysophospholipids were found to be ineffective in stimulating Ca²⁺ spiking, and inhibitors of AA metabolism did not prevent AVP-stimulated Ca²⁺ spiking. Thin layer chromatography was used to monitor the release of AA and phosphatidic acid (PA), which are the products of PLA₂ and phospholipase D (PLD), respectively. AVP (100 pM) stimulated both AA and PA formation, but only PA formation was inhibited by ONO-RS-082 (5 µM). Exogenous PLD (type VII; 2.5 U/ml) stimulated Ca²⁺ spiking equivalent to the effect of 100 pM AVP. AVP stimulated transphosphatidylation of 1-butanol (a PLD-catalyzed reaction) but not 2-butanol, and 1-butanol (but not 2-butanol) completely prevented AVP-stimulated Ca²⁺ spiking. Protein kinase C (PKC) inhibition, which completely prevents AVP-stimulated Ca²⁺ spiking, did not inhibit AVP-stimulated phosphatidylbutanol formation. These results suggest that AVP-stimulated Ca²⁺ spiking depends on activation of PLD rather than PLA₂ and that PKC activation may be downstream of PLD in the signaling cascade.