Serotonin (5-HT) dilates precapillary arterioles in skeletal muscle. The purpose of this study was to determine if 5-HT releases endothelium-derived relaxing factor (EDRF) in this tissue. Diameters of third-order arterioles (A3) in the cremaster muscle of pentobarbital-anesthetized rats were measured via videomicroscopy. Concentration-response curves for acetylcholine, nitroprusside and 5-HT were obtained before and after the application of either hydroquinone (5 x 10(-4) M) or NG-nitro-L-arginine (10(-4) M). The involvement of prostaglandins was eliminated by ibuprofen (10(-4) M). In one group, 5-HT (20 micrograms/kg) and NG-nitro-L-arginine were given i.v. (30 mg/kg). The non-EDRF-dependent vasodilator papaverine (10(-5) M) was applied at the end of the protocol to determine the maximal resting diameter. When applied topically, both hydroquinone and NG-nitro-L-arginine significantly inhibited the dilation induced by acetylcholine, but neither agent affected the dilation to nitroprusside or 5-HT. NG-Nitro-L-arginine (i.v.) attenuated acetylcholine-induced dilation but not the dilation to intravenous 5-HT. These data suggest that 5-HT-induced dilation of small arterioles in skeletal muscle is EDRF-independent.