The aims of this study were to assess the role of nitric oxide (NO) and the contribution of different NO synthase (NOS) isoforms in skeletal muscle contractile dysfunction in septic shock. Four groups of conscious rats were examined. Group 1 served as control; group 2, 3, and 4 were injected with Escherichia coli endotoxin [lipopolysaccharide (LPS), 20 mg/kg i.p.] and killed after 6, 12, and 24 h, respectively. Protein expression was assessed by immunoblotting and immunostaining. LPS injection elicited a transient expression of the inducible NOS isoform, which peaked 12 h after LPS injection and disappeared within 24 h. This expression coincided with a significant increase in nitrotyrosine formation (peroxynitrite foot-print). Muscle expression of the endothelial and neuronal NOS isoforms, by comparison, rose significantly and remained higher than control levels 24 h after LPS injection. In vitro measurement of muscle contractility 24 h after LPS injection showed that incubation with NOS inhibitor (S-methyliosothiourea) restored the decline in submaximal force generation, whereas maximal muscle force remained unaffected. We conclude that NO plays a significant role in muscle contractile dysfunction in septic animals and that increased NO production is due to induction of the inducible NOS isoform and upregulation of constitutive NOS isoforms.