We have examined the role of Stat1α in the induction by LPS of the mouse inducible nitric oxide synthase (EC 1.14. 13.39) gene. LPS induced both the tyrosine phosphorylation of Stat1α and the production of nitric oxide in a time-and dose-dependent manner. The phosphorylation of Stat1α elicited by LPS differed from that observed using IFN-γ or IFN-β, in that LPS induced less phosphorylated protein and the time course of induction was much delayed (2–4 h compared with 30 min). Cycloheximide inhibited LPS-mediated Stat1α phosphorylation. In addition, cell culture supernatants derived from macrophages treated with LPS for 4 h could be transferred to naive macrophage cultures resulting in rapid (30 min), rather than delayed (4 h), phosphorylation of Stat1α. Together, these results implicated an autocrine/paracrine effector protein (s) in the phosphorylation process. LPS stimulated phosphorylation of Stat1α in peritoneal macrophages derived from IFN-γ-knockout mice, negating any possibility that IFN-γ was the mediator. By contrast, neutralizing Ig raised against mouse IFN-αβ inhibited both the delayed LPS-mediated phosphorylation of Stat1α and the rapid induction of phosphorylation induced by supernatants from LPS-stimulated cultures. Collectively, these results show that LPS-induced IFN-αβ production, Stat1α activation, and nitrite accumulation closely parallel one another, suggesting that indirect activation of transcription factor Stat1α by IFN-αβ is a critical determinant of LPS-mediated inducible nitric oxide synthase gene expression.