In this study we tested the hypothesis that lactate, independent of changes in pH, can affect skeletal muscle blood flow through arteriolar dilation that may be mediated by guanosine 3',5'-cyclic monophosphate. Isolated, cannulated, and pressurized first-order rat cremaster skeletal muscle arterioles were studied in a chamber containing Krebs-bicarbonate buffer under no-flow conditions. At pH 7.4 and PO2 of 65 Torr, neutralized lactic acid (lactate) and pyruvic acid (pyruvate) caused arteriolar dilation over the 1-10 mM concentration range. This response to lactate was not altered by 10(-5) M indomethacin, 10(-4) M NG-nitro-L-arginine, or removal of the endothelium. However, responses to 1 and 3 mM pyruvate were significantly inhibited by 100% by endothelium removal, and the response to 10 mM pyruvate was inhibited by 71%. The relaxation of endothelium-denuded arterioles to lactate was inhibited by 10 microM methylene blue, 10 microM LY-83583, hypoxia (PO2 7-10 Torr), and diphenyliodonium, an inhibitor of superoxide-producing flavo-protein enzymes. In contrast, arteriolar dilation to the acidification of the Krebs buffer to pH 7.15, produced by increasing the CO2 concentration of the gas mixture from 5 to 10%, was not inhibited by methylene blue. These results are consistent with lactate-induced skeletal muscle arteriolar dilation being dependent on H2O2-mediated activation of vascular smooth muscle guanylate cyclase and independent of endothelium-derived mediators.