Complete activation of macrophages during immune responses results from stimulation with the activating cytokine interferon‐γ (IFN‐γ) 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‐γ 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‐γ, the pool of molecules phosphorylated on both Tyr701 (Y701) and S727 was strongly increased. Consistently, Stat1‐dependent transcription in response to IFN‐γ 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‐γ. 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‐γ response. The data suggest Stat1 to be a convergence point for immunological stimuli in a macrophage proinflammatory response.