This study was conducted to test the hypothesis that the cytochrome P-450 (CYP450) metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) contributes to the afferent arteriolar response to P2 receptor activation. Afferent arteriolar responses to ATP, the P2X agonist, α,β-methylene ATP and the P2Y agonist UTP were determined before and after treatment with the selective CYP450 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). Stimulation with 1.0 and 10 μM ATP elicited an initial preglomerular vasoconstriction of 12 ± 1% and 45 ± 4% and a sustained vasoconstriction of 11 ± 1% and 11 ± 2%, respectively. DDMS or 20-HEDE significantly attenuated the sustained afferent arteriolar constrictor response to ATP. α,β-Methylene ATP (1 μM) induced a rapid initial afferent vasoconstriction of 64 ± 3%, which partially recovered to a stable diameter 10 ± 1% smaller than control. Both DDMS and 20-HEDE significantly attenuated the initial vasoconstriction and abolished the sustained vasoconstrictor response to α,β-methylene ATP. UTP decreased afferent diameter by 50 ± 5% and 20-HEDE did not change this response. In addition, the ATP-induced increase in the intracellular Ca²⁺ concentration in preglomerular microvascular smooth muscle cells was significantly attenuated by 20-HEDE. Taken together, these results are consistent with the hypothesis that the CYP450 metabolite 20-HETE participates in the afferent arteriolar response to activation of P2X receptors.