We determined whether salicylate at pharmacological concentrations inhibits nitric-oxide synthase-2 (NOS-2) and cyclo-oxygenase-2 (COX-2) expressions in RAW 264.7 stimulated with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). Cells were treated with sodium salicylate (10(-7)-10(-4) m) or vehicle for 30 min followed by LPS+IFN-gamma for up to 24 h. Salicylate suppressed NOS-2 and COX-2 protein levels and promoter activities stimulated by LPS+IFN-gamma for 4 h in a concentration-dependent manner but had no effect on NOS-2 expression stimulated by the combined agonists for 24 h. Results from promoter analysis indicate that the binding of CCAAT/enhancer-binding protein beta (C/EBPbeta) to its cognate site at -150/-142 on the NOS-2 promoter region was essential for NOS-2 expression at 4 h but not at 24 h. Salicylate reduced C/EBPbeta binding at 4 h and did not alter its binding at 24 h. NOS-2 and COX-2 protein levels and C/EBPbeta binding stimulated by LPS+IFN-gamma for 4 h were inhibited by a similar battery of signaling inhibitors, suggesting a common pathway for NOS-2 and COX-2 expression. Kinetic analysis indicates that NOS-2, similar to COX-2 expression, at 4 h was largely due to the action of LPS, which induced C/EBPbeta binding, whereas its expression at a longer time point was contributed by IFN-gamma. Our findings implicate two distinct pathways for NOS-2 expression induced by LPS+IFN-gamma. Salicylate at pharmacological concentrations is capable of suppressing the early phase of NOS-2 and COX-2 expression by blocking C/EBPbeta binding.