Complete activation of macrophages during immune responses results from stimulation with the activating cytokine interferon-gamma (IFN-gamma) and a second stimulus, usually a microbial product. Bacterial infection of macrophages, or treatment with bacterial lipopolysaccharide (LPS), resulted in rapid Stat1 phosphorylation on Ser727 (S727) independently of concomitant tyrosine phosphorylation. IFN-gamma also caused rapid phosphorylation of S727. In both situations, S727 phosphorylation was reduced by pre-treatment of cells with the serine kinase inhibitor H7. When macrophages were treated sequentially or simultaneously with LPS and IFN-gamma, the pool of molecules phosphorylated on both Tyr701 (Y701) and S727 was strongly increased. Consistently, Stat1-dependent transcription in response to IFN-gamma was significantly enhanced if the cells were pre-treated with bacterial LPS. The relative amount of S727-phosphorylated Stat1 in the non-tyrosine phosphorylated fraction was considerably smaller than that in the tyrosine-phosphorylated fraction. No evidence was found for an effect of S727 phosphorylation on the phosphorylation of Y701 by IFN-gamma. Thus, serine and tyrosine phosphorylation of Stat1 are caused independently of each other, but the serine kinase may recognize tyrosine-phosphorylated Stat1 preferentially in the course of an IFN-gamma response. The data suggest Stat1 to be a convergence point for immunological stimuli in a macrophage proinflammatory response.