The kidney plays a central role in long-term regulation of arterial blood pressure and salt and water homeostasis. This is achieved in part by the local actions of paracrine and autacoid mediators such as the arachidonic acid-prostanoid system. The present study tested the role of specific PGE₂ E-prostanoid (EP) receptors in the regulation of renal hemodynamics and vascular reactivity to PGE₂. Specifically, we determined the extent to which the EP₂ and EP₃ receptor subtypes mediate the actions of PGE₂ on renal vascular tone. Renal blood flow (RBF) was measured by ultrasonic flowmetry, whereas vasoactive agents were injected directly into the renal artery of male mice. Studies were performed on two independent mouse lines lacking either EP₂or EP₃ (−/−) receptors and the results were compared with wild-type controls (+/+). Our results do not support a unique role of the EP₂ receptor in regulating overall renal hemodynamics. Baseline renal hemodynamics in EP₂−/− mice [RBF EP₂−/−: 5.3 ± 0.8 ml · min⁻¹ · 100 g kidney wt⁻¹; renal vascular resistance (RVR) 19.7 ± 3.6 mmHg · ml⁻¹ · min · g kidney wt] did not differ statistically from control mice (RBF +/+: 4.0 ± 0.5 ml · min⁻¹ · 100 g kidney wt⁻¹; RVR +/+: 25.4 ± 4.9 mmHg · ml⁻¹ · min · 100 g kidney wt⁻¹). This was also the case for the peak RBF increase after local PGE₂ (500 ng) injection into the renal artery (EP₂−/−: 116 ± 4 vs. +/+: 112 ± 2% baseline RBF). In contrast, we found that the absence of EP₃receptors in EP₃−/− mice caused a significant increase (43%) in basal RBF (7.9 ± 0.8 ml · min⁻¹ · g kidney wt⁻¹,P < 0.05 vs. +/+) and a significant decrease (41%) in resting RVR (11.6 ± 1.4 mmHg · ml⁻¹ · min · g kidney wt⁻¹, P < 0.05 vs. +/+). Local administration of 500 ng of PGE₂ into the renal artery caused more pronounced renal vasodilation in EP₃−/− mice (128 ± 2% of basal RBF, P < 0.05 vs.+/+). We conclude that EP₃ receptors mediate vasoconstriction in the kidney of male mice and its actions are tonically active in the basal state. Furthermore, EP₃receptors are capable of buffering PGE₂-mediated renal vasodilation.