Inhibition of nitric oxide synthase (NOS) activity in vivo impedes hypertrophy in the overloaded rat plantaris. We investigated the mechanism for this effect by examining early events leading to muscle growth following 5 or 12d of functional overload. Male Sprague-Dawley rats (~350g) were randomly divided into three treatment groups: Control, L-NAME (N-nitro-L-arginine methyl ester; 90mg/kg/d), and TRIM (1-(2-trifluoromethyl-phenyl)-imidazole; 10mg/kg/d). Unilateral removal of synergists induced chronic overload (OL) of the right plantaris. Sham surgery performed on the left hindlimb served as a normally loaded (NL) control. L-NAME and TRIM treatments prevented OL-induced skeletal -actin and type I (slow) myosin heavy chain(MHC) mRNA expression at 5d. Conversely, neither L-NAME nor TRIM affected hepatocyte growth factor (HGF) or vascular endothelial growth factor (VEGF) mRNA responses to OL at 5d. However, OL-induction of insulin-like growth factor-1 (IGF-1) and mechano growth factor (MGF) mRNA was greater (P<0.05) in the TRIM group compared to the Controls. Further, the phosphorylated/total p70 s6 kinase (p70^s6K) ratio was higher in OL muscle from NOS-inhibited groups, compared to Control-OL. At 12d of OL, the cumulative proliferation of plantaris satellite cells was assessed by subcutaneous implantation of time-release BrdU pellets during the overload-inducing surgeries. Although OL caused a 5-fold increase in the number of mitotically active (BrdU+) sub-laminar nuclei, this was unaffected by concurrent NOS inhibition. Therefore, NOS activity may provide negative feedback control of IGF-1/ p70^s6K signaling during muscle growth. Moreover, NOS activity may be involved in transcriptional regulation of skeletal -actin and type I (slow) MHC during functional overload.