Because the induction of interleukin-1β (IL-1β) is critical to antibacterial host defenses and its excessive generation is a prominent component of sepsis, regulation of this proinflammatory cytokine is a critical factor in the immune response to lipopolysaccharide (LPS). We previously showed that LPS-induced IL-1β expression was regulated by a Stat1-dependent, nitric oxide (NO)-mediated mechanism. Subsequent in vivo studies showed that whereas Stat1 had a role in the downregulation of IL-1β expression, it had a more significant effect on its initial induction. Although both interferon-β (IFN-β) and IFN-γ activate Stat1, the early appearance of IFN-β in the circulation after LPS administration suggested its pivotal role in Stat1-mediated IL-1β expression in vivo. Further in vitro analysis of peritoneal macrophages from IFN-β ^–/–, Stat1^–/–, and caspase-1^–/– mice and their wild-type controls following LPS stimulation demonstrated that IL-1β mRNA was expressed in these mice but not in macrophages from MyD88^–/– mice. Despite the presence of IL-1β mRNA, IL-1β protein was markedly reduced in the absence of Stat1 activation in macrophages derived from IFN-β ^–/– and Stat1^–/– mice or in the absence of caspase-1 activity, which itself was dependent on Stat1 activation. These studies support the hypothesis that the expression of IL-1β requires both the MyD88-dependent induction of IL-1β mRNA and pro-IL-1β as well as the MyD88-independent, Stat1-mediated processing of that gene product into active cytokine.