1. The effects of the nitric oxide (NO) donor sodium nitroprusside (SNP) on the rates of glucose transport and utilization and its interaction with insulin were investigated in rat soleus muscle in vitro. SNP stimulated the rate of 2-deoxyglucose transport and insulin-mediated (100 Ɓ-units/ml) rates of both net and [¹⁴C]lactate release and the rate of glucose oxidation. The effects of SNP were independent of the concentration-dependent effects of insulin on glucose metabolism. 2. SNP stimulated the insulin-stimulated rates of net and [¹⁴C]lactate release and glucose oxidation in a concentration-dependent manner. The rate of [¹⁴C]lactate release was also stimulated by another NO donor, (Z)-1-(N-[aminopropyl]-N-[4-(3-aminopropylammonio)butyl]-amino)-diazen-1-ium-1,2-diolate (spermine NONOate). 3. SNP at 5, 10 and 15 mM inhibited the insulin-stimulated rate of glycogen synthesis and this rate was further decreased at 20 and 25 mM SNP. SNP did not affect the rate of glycogen synthesis in the absence of insulin. 4. Haemoglobin, which is a NO scavenger, prevented the stimulation of the rates of [¹⁴C]lactate release by SNP or spermine NONOate. 5. The cGMP content was increased maximally (by approx. 80-fold) within 15 min by SNP (15 mM). The cGMP content, raised maximally by SNP, was significantly decreased by the guanylate cyclase inhibitor LY-83583 (10 ƁM). The cGMP analogue 8-bromo-cGMP (100 ƁM) significantly increased the rate of net lactate release. 6. LY-83583 significantly inhibited SNP-stimulated rates of 2-deoxyglucose transport, [¹⁴C]lactate release and glucose oxidation. Methylene Blue (another guanylate cyclase inhibitor) also inhibited SNP-stimulated rates of [¹⁴C]lactate release. 7. The results suggest that in rat skeletal muscle: (a) nitric oxide (from SNP or spermine NONOate) increases the rate of glucose transport and metabolism, an effect independent of insulin; (b) SNP inhibits insulin-mediated rates of glycogen synthesis; (c) SNP stimulates cGMP formation, which mediates, at least partly, the effects on glucose metabolism; (d) nitric oxide-mediated stimulation of glucose utilization might occur in fibre contraction. The implications of the effects of NO on glucose metabolism are discussed.