Skeletal muscle arterioles are known to constrict upon elevation of ambient PO2. While several studies have shown that the endothelium plays an important role in this response. it is not clear how this response is mediated. We examined the oxygen-induced constriction of arterioles in the rat spinotrapezius muscle. Elevation of superfusion solution PO2 from about 15 to 150 mm Hg caused arteriolar constriction by 25% (±3%, n = 18). Inhibition of prostaglandin synthesis by superfusion of indomethacin (30 μM) produced vasoconstriction by 28% (±9.5%, n = 5), but left the PO2 response unaffected. Blockade of the synthesis of endothelium-derived relaxing factor (EDRF) by NG-nitro-L-arginine (L-NNA, 35 mg/kg iv) caused arteriolar constriction by 31% (±8%, n = 8). During application of L-NNA, the constrictor response to PO2 elevation was reduced to 3 ± 2%. Administration of superoxide dismutase (SOD, 80,000 U/kg iv) did not affect the PO2 response. It is concluded that in small arterioles of skeletal muscle both EDRF and prostanoids sustain a significant basal dilatation. The dilatory effects of EDRF but not of prostaglandins are strongly dependent on PO2. The vasoconstriction in response to high ambient PO2 is not due to EDRF breakdown during its diffusion from endothelial to smooth muscle cells.