This study investigated the dosage effects of nitric oxide synthase (NOS) inhibitor L-NMMA on intermittent pneumatic compression (IPC)-induced vasodilation in uncompressed upstream muscle, and the effects of IPC on endothelial NOS (eNOS) expression in upstream muscle. Following L-NMMA infusion, mean arterial pressure (MAP) increased by 5% from baseline (99.5 ± 18.7 mm Hg) (p<0.05). Heart rate and respiratory rate were not significantly affected. One hour IPC application on legs induced 10% dilation from baseline in 10-20 µm arterioles and 10-20% dilation in 21-40 µm arterioles and 41-70 µm arteries in uncompressed cremaster muscle. IPC-induced vasodilation was dose-dependently reduced, abolished, or even reversed by concurrently infused L-NMMA. Moreover, the expression of eNOS mRNA in uncompressed cremaster muscle was upregulated to 2- and 2.5-times normal at the end of 1h and 5 h IPC on legs, respectively, and the expression of eNOS protein was upregulated to 1.8-times normal. These increases returned to baseline level after cessation of IPC. The results suggest that eNOS plays an important role in regulating the microcirculation in upstream muscle during IPC.