The uptake and metabolism of glucose by skeletal muscle is a major determinant of whole body glucose homeostasis. Contraction is a strong stimulus of muscle glucose uptake that still occurs in insulin-resistant muscle. The intramuscular signaling pathways associated with contraction-mediated muscle glucose uptake are not fully understood but are separate from insulin signaling. The effects of NOS inhibition on contraction-mediated glucose uptake have yielded contradictory results due to systemic effects of NOS inhibition and measurements being made after rather than during contraction. In this study we examined the effect of local infusion of L-NAME (5µM) into one rat hindleg via the epigastric artery during muscle contraction (35V field stimulation, 0.1 msec pulses at 2Hz). Measurements included systemic blood pressure, heart rate, muscle contraction force, femoral blood flow, capillary recruitment (by contrast-enhanced ultrasound), muscle glucose uptake (by ³H-2-deoxyglucose uptake), muscle NOS activity, muscle nitrate and nitrite content (NOx), and AMPK signaling (AMPKα Thr 172 phosphorylation, ACCβ Ser222 phosphorylation and nNOS Ser 1412 phosphorylation). Local L-NAME infusion had no effect on blood pressure or heart rate indicating there were no systemic actions. The contraction-mediated increase in NOS activity and NOx content in the L-NAME treated leg were reversed while the nNOS phosphorylation remained increased. Since, NOS activity measurements from muscle at the end of the experiments were made in vitro in the absence of L-NAME, the results suggest that L-NAME in vivo has other actions than simply acting as a competitive inhibitor of NOS. The contraction-mediated increase in AMPKα and ACCβ phosphorylation, and muscle contractile force were unaffected by L-NAME infusion. L-NAME caused a significant (P<0.05) decrease (30%) in femoral blood flow but not capillary recruitment during contraction and significantly (P<0.001) reduced the contraction-mediated glucose uptake by (32%). In conclusion, NOS inhibition attenuated increases in skeletal muscle glucose uptake without influencing capillary recruitment or AMPK signaling. This suggests that nitric oxide is independently involved in the signaling pathway for contraction-mediated skeletal muscle glucose uptake distal to glucose delivery. [ABSTRACT FROM AUTHOR]