Activation of TLRs is most closely associated with induction of pro-inflammatory gene expression; however, expression of many other genes, including the TLR genes themselves, has also been shown to be modulated following TLR engagement. A large family of nuclear transcription factors, the interferon regulatory factors (IRFs), have been implicated in TLR signaling leading to pro-inflammatory gene expression. Given that IRF-1 and IRF-2 counter-regulate the transcriptional activity of many genes, we hypothesized that IRF-1 and IRF-2 might also regulate TLR gene expression following LPS stimulation of murine macrophages. mRNA derived from medium- or LPS-treated primary peritoneal macrophages was analyzed for TLR gene expression using quantitative real-time PCR. In wild-type macrophages, LPS up-regulated expression of TLRs 1-3 and 6-9 steady-state mRNA, while TLR4 mRNA was modestly down-regulated. IRF-2(-/-) macrophages responded to LPS with dysregulated expression of TLR3, TLR4, and TLR5 mRNA, whereas IRF-1 deficiency dampened LPS-induced mRNA expression for TLR3, TLR6, and TLR9. Functional studies revealed aberrant TLR3 signaling in IRF-2(-/-) macrophages. Collectively, these findings reveal an additional level of complexity associated with TLR transcriptional regulation and suggest that the trans-acting factors, IRF-1 and IRF-2, contribute to the innate immune response to infections by regulating TLR gene expression.