Background: Both nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) are established as important factors determining the vascular tone. The relative contribution of these factors to the renal microvascular tone, however, has not been delineated. Methods: Isolated perfused hydronephrotic rat kidneys were used to characterize the relative role of NO and EDHF in mediating the tone of interlobular arteries (ILA) and afferent arterioles (AFF). Results: During the norepinephrine constriction, acetylcholine (ACH, 1 µmol/l) induced a sustained vasodilation of ILA (90 ± 9% reversal) and AFF (117 ± 13% reversal). In the presence of nitro- L-arginine methylester (LNAME), the ACH-induced vasodilation of ILA and AFF was converted to transient dilation, with only 53 ± 7 and 32 ± 7% reversal observed 10 min after 1 µmol/l ACH (i.e sustained phase). In contrast, LNAME had no effect on the initial phase of ACH-induced dilation. In the presence of apamin + charybdotoxin, the initial vasodilator response to ACH (1 µmol/l) was diminished (ILA, from 108 ± 8 to 46 ± 9%; AFF, from 108 ± 14 to 58 ± 8%), whereas no impairment was observed in sustained phases. Furthermore, the magnitude of the vasoconstriction caused by LNAME was greater at smaller vessel segments. Finally, the LNAME-induced inhibition of the sustained phase of ACH-induced vasodilation was greater as the vessel diameter decreased. Conclusions: That the relative contribution of NO and EDHF differs, with a greater role of NO in the basal tone and ACH-induced vasodilation at smaller vascular segments of ILA and AFF.