This study tested the hypothesis that P2X receptor activation increases intracellular Ca 2+ concentration ([Ca 2+] i) in preglomerular microvascular smooth muscle cells (MVSMC) by evoking voltage-dependent calcium influx. MVSMC were obtained and loaded with the calcium-sensitive dye fura 2 and studied by using single-cell fluorescence microscopy. The effect of P2X receptor activation on [Ca 2+] i was assessed by using the P2X receptor-selective agonist α,β-methylene-ATP and was compared with responses elicited by the endogenous P2 receptor agonist ATP. α,β-Methylene-ATP increased [Ca 2+] i dose dependently. Peak increases in [Ca 2+] i averaged 37 ± 11, 73 ± 15, and 103 ± 21 nM at agonist concentrations of 0.1, 1, and 10 μM, respectively. The average peak response elicited by 10 μM α,β-methylene-ATP was ∼34% of the response obtained with 10 μM ATP. α,β-Methylene-ATP induced a transient increase in [Ca 2+] i before [Ca 2+] i returned to baseline, whereas ATP induced a biphasic response including a peak response followed by a sustained plateau. In Ca 2+-free medium, ATP induced a sharp transient increase in [Ca 2+] i, whereas the response to α,β-methylene-ATP was abolished. Ca 2+ channel blockade with 10 μM diltiazem or nifedipine attenuated the response to α,β-methylene-ATP, whereas nonspecific blockade of Ca 2+ influx pathways with 5 mM Ni 2+ abolished the response. Blockade of P2X receptors with the novel P2X receptor antagonist NF-279 completely but reversibly abolished the response to α,β-methylene-ATP. These results indicate that P2X receptor activation by α,β-methylene-ATP increases [Ca 2+] i in preglomerular MVSMC, in part, by stimulating voltage-dependent Ca 2+ influx through L-type Ca 2+ channels.