The inducible form of nitric oxide synthase (NOS2) catalyzes the synthesis of nitric oxide (NO) from arginine in response to injury and infection. NOS2 is expressed predominantly by macrophages and lymphocytes. However, skeletal muscle also expresses NOS2 in response to inflammatory stimuli. The present study sought to determine whether lipopolysaccharide (LPS) stimulates NOS2 in skeletal muscle via Toll-like receptor-4 (TLR4). Intraperitoneal injection of LPS in wild-type mice (C3H/HeSnJ) increased NOS2 mRNA fourfold in skeletal muscle, while no change in NOS2 mRNA was observed in C3H/HeJ mice that harbored a mutation in the LPS receptor. NOS2 coimmunoprecipitated with the muscle-specific caveolin-3 protein, suggesting that myofibers per se respond to LPS in vivo. LPS stimulated NOS2 mRNA expression in C₂C₁₂ myocytes, and the regulation of NOS2 mRNA was comparable in myoblasts and differentiated myotubes. LPS transiently stimulated the phosphorylation of the interleukin-1 receptor-associated kinase (IRAK-1) in C₂C₁₂ cells and decreased the total amount of IRAK-1 both in vitro and in vivo over time. LPS stimulated the expression of an NF-κβ reporter plasmid, and this was inhibited by the proteasomal inhibitor MG-132. Both myoblasts and myotubes expressed TLR2 and TLR4 mRNA. Expression of a dominant negative form of TLR4 in C₂C₁₂ cells blocked LPS-induced NF-κβ reporter activity. SP-600125 [a c-Jun NH₂-terminal kinase (JNK) inhibitor] also prevented LPS stimulation of NOS2 expression. Moreover, the JNK inhibitor prevented the LPS-induced increase in NO synthesis. These data indicate that LPS increases NOS2 mRNA expression in muscle via a TLR4-dependent mechanism.