The in vitro blood-perfused juxtamedullary nephron technique was utilized to determine the contribution of endothelium-derived relaxing factor (EDRF) to resting renal arteriolar caliber and to evaluate the interaction between EDRF and angiotensin II (ANG II) in renal microvascular control. Video microscopy was employed to visualize rat afferent and efferent arterioles and to measure their responses to blockade of nitric oxide (NO), which has been shown to account for much of the biological action of EDRF. The NO synthesis inhibitor, Nω-nitro-L-arginine (L-NNA), elicited vasoconstriction in a concentration-dependent manner, with 1,000 μM L-NNA significantly reducing both afferent (16 ± 3%) and efferent (13 ± 1%) diameters. This concentration of L-NNA also blocked the vasodilator response to 10 μM acetylcholine, while responsiveness to sodium nitroprusside was maintained. Vasoconstrictor responses to 1,000 μM L-NNA were attenuated in kidneys from rats pretreated with enalaprilat or losartan, reducing afferent diameter by 7 ± 1 (n = 8) and 3 ± 1% (n = 10) of control, respectively. Efferent arteriolar responses to L-NNA were similarly attenuated by losartan. The constrictor response to 10 nM ANG II was not exaggerated by L-NNA, suggesting that ANG II does not stimulate EDRF synthesis. These observations indicate that EDRF is continuously released in a quantity sufficient to affect both afferent and efferent arterioles of juxtamedullary nephrons in vitro. Furthermore, ANG II blockade attenuates the vasoconstriction elicited by L-NNA, suggesting that EDRF interacts with the renin-angiotensin system to control juxtamedullary afferent and efferent arteriolar resistance. © 1992 the American Physiological Society.