The current study tested the hypothesis that endogenous 20-hydroxyeicosatetraenoic acid (20-HETE) contributes to the increase in intracellular calcium ([Ca2+]i) elicited by P2X receptor activation in renal microvascular smooth muscle cells. Vascular smooth muscle cells obtained from rats were loaded with fura-2 and studied using standard single cell fluorescence microscopy. Basal renal myocyte [Ca2+] i averaged 96 ± 5 nM. ATP (10 and 100 μM) increased vascular smooth muscle cell [Ca2+]i by 340 ± 88 and 555 ± 80 nM, respectively. The cytochrome P450 hydroxylase inhibitor, N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), or the 20-HETE antagonist, 20-hydroxyeicosa-6(Z), 15(Z)-dienoic acid (20-HEDE), significantly attenuated the peak myocyte [Ca2+]i responses to 10 and 100 μM ATP. ATP (100 μM) increased vascular smooth muscle cell [Ca 2+]i by 372 ± 93 and 163 ± 55 nM in the presence of DDMS or 20-HEDE, respectively. The P2X receptor agonist, α,β-methyleneylene-ATP (10 μM), increased myocyte [Ca 2+]i by 78 ± 12 nM, and this response was significantly attenuated by DDMS (40 ± 15 nM). In contrast, the vascular smooth muscle cell [Ca2+]i evoked by the P2Y agonist, UTP (100 μM), was not altered by DDMS or 20-HEDE. The effect of 20-HETE on [Ca2+]i was also assessed, and the peak increases in [Ca2+]i averaged 62 ± 12 and 146 ± 70 nM at 20-HETE concentrations of 1 and 10 μM, respectively. These results demonstrate that 20-HETE plays a significant role in the renal microvascular smooth muscle cell [Ca2+]i response to P2X receptor activation.