This study examined the effects of acute exercise on skeletal muscle nitric oxide synthase (NOS) activity. Female Sprague-Dawley rats were divided into three groups: control, exercise, and exercise + N(G)-nitro-L-arginine methyl ester (L-NAME). In the exercise + L-NAME group, L-NAME was administered in the drinking water (1 mg/ml) for 2 days and subsequently the exercise and exercise + L-NAME groups underwent a 45-min bout of exhaustive treadmill running after which NOS activity and muscle glycogen were measured. In the control and exercise groups, 1-amino-S-methylisothiourea (AMITU), a selective neuronal NOS inhibitor, with and without additional nonselective NOS blockade [with N(G)-monomethyl-L-arginine (L-NMMA)], was used in vitro to assess the contribution of nNOS to total NOS activity. The exercise bout increased NOS activity by 37% in exercise compared with control groups, and both groups had significantly greater NOS activity compared with exercise + L-NAME. AMITU decreased total NOS activity in the control and exercise groups by 31.8 and 30.2%, respectively, and these activities were significantly greater than AMITU + L-NMMA in both control and exercise groups. We conclude that 1) there is basal neuronal NOS and endothelial NOS activity in skeletal muscle, 2) an acute exercise bout increases NOS activity in skeletal muscle, and 3) glycogen depletion during exercise occurs irrespective of NOS activity.