To determine the role of C-Rel in nitric-oxide synthase-2 (NOS-2) transcriptional activation, we evaluated the effect of lipopolysaccharide and interferon-gamma (LPS/IFNgamma) on C-Rel DNA binding in RAW 264.7. LPS/IFNgamma-stimulated C-Rel binding peaked at 4 to 8 h and declined at 24 h. Transfection of cells with a C-Rel small interfering RNA abrogated C-Rel binding at all time points. LPS/IFNgamma produced superoxide at 4 h, which subsided at 8 h. C-Rel binding and NOS-2 expression were abrogated by superoxide dismutase or apocynin at 4 h, suggesting a key role that superoxide plays in mediating C-Rel binding and NOS-2 transactivation only at 4 h. We have reported previously that salicylate at 10(-5) M inhibited LPS/IFNgamma-induced CCAAT/enhancer binding protein beta (C/EBPbeta) binding at 4 h but not at 8 or 24 h. A single dose of salicylate did not inhibit C-Rel binding at any time point. The addition of a second dose of salicylate 4 h before an indicated endpoint suppressed C-Rel but not C/EBPbeta or interferon-gamma-regulated factor-1 binding at 8 and 24 h. A single dose of salicylate added with LPS/IFNgamma inhibited NOS-2 expression only at 4 h. However, salicylate supplement inhibited NOS-2 promoter activities and mRNA and protein levels throughout 24 h. Signal profiling with a panel of inhibitors revealed time-dependent switch of signaling pathways. These results demonstrate temporal regulation of transactivator binding by LPS/IFNgamma via evolving signaling pathways. We propose that salicylate inhibits C/EBPbeta binding at 4 h and C-Rel binding at 8 and 24 h by targeting related kinases.