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Nitric oxide synthases (NOS) convert L-arginine to nitric oxide in the presence of tetrahydrobiopterin (BH4). Two constitutive isoforms of NOS exist in normal skeletal muscle fibers, however, the existence of a third, the inducible isoform (iNOS), has never been detected in these fibers in vivo. Therefore, we assessed the influence of in vivo endotoxemia on skeletal muscle expression of constitutive and inducible NOS isoforms and GTP cyclohydrolase I, the rate limiting enzyme of BH4 synthesis. Two groups of rats were infused i.p. either with E. coli endotoxin (20 mg/kg, LPS group) or saline (saline group). Animals were killed 6 h later and the ventilatory and limb muscles were quickly frozen. Endotoxin infusion elicited a significant rise in NOS activity of the diaphragm, intercostal, soleus and gastrocnemius muscles. Reverse transcription-polymerase chain reaction (RT-PCR) on muscle total RNA detected very low expression of iNOS and GTP cyclohydrolase I mRNA in the saline group, but significant upregulation of both enzymes was found in the ventilatory and limb muscles of the LPS group. Immunoblotting detected no iNOS protein in the saline group but a significant iNOS protein expression was found in the diaphragm, intercostal and soleus muscles and to a lesser extent, in the gastrocnemius of the LPS group. Endotoxemia was also associated with increased protein expression of constitutive NOS isoforms mainly in the diaphragm and to lesser extent in the intercostal, gastrocnemius and soleus muscles. We conclude that in vivo exposure to endotoxin leads to differential induction of both iNOS and GTP cyclohydrolase I in the ventilatory and limb muscles.