The effects of salicylate on the phosphorylation and nuclear translocation of signal transducers and activators of transcription (STATs) induced by interferon-gamma (IFN-gamma) were studied in rat cardiac fibroblasts as a possible model for the anti-inflammatory effects of salicylate on this signaling pathway. Salicylate inhibited the tyrosine phosphorylation of both STAT1 and STAT3, but had a more pronounced effect on STAT3 activation. Salicylate pretreatment prevented both the nuclear translocation and the DNA-binding activity of STAT1 and STAT3, assessed by immunoblotting and gel shift assays, respectively. In addition to causing phosphorylation at tyrosine residues, IFN-gamma also phosphorylated STAT3 and STAT1 at serine 727. Salicylate attenuated both tyrosine and serine phosphorylations of STAT3, and also suppressed extracellular signal-regulated kinase (ERK) activation, implicating the effect of salicylate on ERK as a possible mechanism for attenuating STAT3 activation. The possibility that salicylate might affect signaling cascades by altering the redox state of the cells was examined, and its effects differed from those of other reducing agents. Salicylate did attenuate the effects of hydrogen peroxide on STAT phosphorylation, consistent with a mechanism involving an interaction between salicylate and reactive oxygen species within the cell.