We investigated the effects of the endothelium-derived nitric oxide system on renal hemodynamics and function during the 3rd trimester in a chronically catheterized fetal sheep preparation. Acetylcholine caused a significant decrease in renal vascular resistance (60% of the baseline value) as compared with aortic constriction (142% of the baseline value). The effects of acetylcholine could be blocked by prior administration of N omega-nitro-L-arginine (renal vascular resistance = 102% of baseline). Sodium nitroprusside also caused a significant drop in renal vascular resistance (63% of baseline), but this could not be blocked by N omega-nitro-L-arginine (77% of baseline). Infusion of N omega-nitro-L-arginine with blood pressure maintained at a constant level resulted in a significant increase in renal vascular resistance (148% of the baseline value) as compared with saline alone (94% of baseline). Glomerular filtration rate increased after saline infusion (156% of the baseline value), but this increase was blocked by N omega-nitro-L-arginine (87% of baseline). Sodium excretion also increased (340%), and this increase was blunted by N omega-nitro-L-arginine (235%). We conclude that basal production of endothelium-derived nitric oxide results in ongoing renal vasodilation in 3rd-trimester fetal sheep, maintaining baseline renal blood flow. The endothelium-derived nitric oxide system can also be stimulated to an increased level of activity, and its blockade partially prevents the homeostatic response of the fetus to volume and salt overload.