Sepsis is a severe catabolic condition. The loss of skeletal muscle protein mass is characterized by enhanced release of the amino acids glutamine and arginine, which (in)directly affects interorgan arginine and the related nitric oxide (NO) synthesis. To establish whether changes in muscle amino acid and protein kinetics are regulated by NO synthesized by NOS2 or NOS3, we studied C57BL6/J wild type (WT), NOS2-deficient (NOS2^-/-), and NOS3-deficient (NOS3^-/-) mice under control (unstimulated) and LPS-treated conditions. Muscle amino acid metabolism was studied across the hindquarter by infusing the stable isotopes L-[phenyl-²H₅]Phenylalanine, L-[phenyl-²H₂]Tyrosine, L-[guanidine-¹⁵N₂]Arginine, and L-[ureido-¹³C; ²H₂]Citrulline. Muscle blood flow was measured using radioactive PAH dilution. Under baseline conditions, muscle blood flow was halved in NOS2^-/- mice (P<0.1), with simultaneous reductions in muscle glutamine, glycine, alanine, and arginine release and glutamic acid, citrulline, valine, and leucine uptake (P<0.1). After LPS treatment, (net) muscle protein synthesis increased in WT and NOS2^-/- mice (LPS vs. control (mean ± SEM): 13 ± 3 vs. 8 ± 1 nmol/10g/min (WT), 18 ± 5 vs. 7 ± 2 nmol/10g/min (NOS2^-/-); P<0.05 for LPS vs. control). This response was absent in NOS3^-/- mice (LPS vs. control: 11 ± 4 vs. 10 ± 2 nmol/10g/min). In agreement, the increase in muscle arginine turnover after LPS was also absent in NOS3^-/- mice. In conclusion, disruption of the NOS2 gene compromises muscle glutamine release and muscle blood flow in control mice, but had only minor effects after LPS. NOS3 activity is crucial for the increase in muscle arginine and protein turnover during early endotoxemia.