The high-af®nity receptor for IgG (FcgRI) is a myeloid cell-speci®c and IFN-g-induced gene, and thereby serves as a paradigm for cytokine-induced cell type-speci®c gene responses. The expression of FcgRI is regulated by PU.1 and Stat1 transcription factors. We established an experimental model to analyze the individual functions of Stat1 and PU.1 in cytokine-induced transcription of the natural FcgRI promoter in U3A cells lacking both factors. PU.1 was required for both the basal activity and for the IFN-g-induced FcgRI promoter activation, while Stat1 alone could not initiate transcription. In contrast, in the context of a heterologous promoter, PU.1 inhibited the Stat1-mediated transcription. Systematic analysis of Stat1 and PU.1 mutants and FcgRI promoter elements revealed that activation of the promoter required the DNA binding, and the transactivation functions of both Stat1 and PU.1. PU.1 and Stat1 bound the promoter elements independently, and no physical interaction between the proteins was observed. The requirement of PU.1 for FcgRI promoter activity was supported by demonstration of in vitro interaction between PU.1 and components of the basal transcription machinery TBP and RNA polymerase II. Deletion of the acidic transactivation domain of PU.1 greatly diminished both the FcgRI promoter activity as well as the interaction with RNA polymerase II. In contrast, Stat1 did not interact with TBP or RNA polymerase II. These results de®ne functional cooperativity between PU.1 and Stat1 in FcgRI promoter activation where PU.1 serves as an ampli®er and bridging factor with the basal transcription machinery.