Cluster of differentiation (CD)-40 is a cell surface receptor belonging to the tumor necrosis factor receptor family that plays a critical role in the regulation of immune responses. We have previously shown that the cytokine interferon (IFN)-γ induces CD40 expression in microglia. Herein, we have elucidated the molecular mechanisms underlying IFN-γ induction of CD40 gene expression in microglia/macrophages. IFN-γ up-regulates CD40 expression at the transcriptional level, and this regulation involves the STAT-1α transcription factor. Microglia from STAT-1α-deficient mice were refractive to IFN-γ induction of CD40 expression, illustrating the importance of STAT-1α in this response. Functional analysis of the CD40 promoter indicates that two gamma activated sequence elements as well as two Ets elements are involved in IFN-γ induction of CD40 promoter activity. STAT-1α binds to the gamma activated sequence elements, whereas PU.1 and/or Spi-B bind to the Ets elements. The expression of PU.1 and Spi-B, in conjuction with STAT-1α activation, correlates with IFN-γ inducibility of CD40 expression. Collectively, our data demonstrate the involvement of STAT-1α, PU.1, and Spi-B in IFN-γ induction of CD40 gene expression in cells of the macrophage lineage.