Macrophages are activated by unmethylated CpG-containing DNA (CpG DNA) via TLR9. IFN-gamma and LPS can synergize with CpG DNA to enhance proinflammatory responses in murine macrophages. Here, we show that LPS and IFN-gamma up-regulated Tlr9 mRNA in murine bone marrow-derived macrophages (BMM). The ability of LPS and IFN-gamma to induce Tlr9 mRNA expression in BMM was dependent on the presence of the growth factor, CSF-1, which is constitutively present in vivo. However, there were clear differences in mechanisms of Tlr9 mRNA induction. LPS stimulation rapidly removed the CSF-1 receptor (CSF-1R) from the cell surface, thereby blocking CSF-1-mediated transcriptional repression and indirectly inducing Tlr9 mRNA expression. By contrast, IFN-gamma activated the Tlr9 promoter directly and only marginally affected cell surface CSF-1R expression. An approximately 100-bp proximal promoter of the murine Tlr9 gene was sufficient to confer basal and IFN-gamma-inducible expression in RAW264.7 cells. A composite IFN regulatory factor (IRF)/PU.1 site upon the major transcription start site was identified. Mutation of the binding sites for PU.1 or IRF impaired basal promoter activity, but only the IRF-binding site was required for IFN-gamma induction. The mRNA expression of the IRF family member IFN consensus-binding protein [(ICSBP)/IRF8] was coregulated with Tlr9 in macrophages, and constitutive and IFN-gamma-inducible Tlr9 mRNA expression was reduced in ICSBP-deficient BMM. This study therefore characterizes the regulation of mouse Tlr9 expression and defines a molecular mechanism by which IFN-gamma amplifies mouse macrophage responses to CpG DNA.