IFN regulatory factor 4 (IRF4) and IFN consensus sequence binding protein (ICSBP) are highly homologous members of the growing family of IRF proteins. ICSBP expression is restricted to lymphoid and myeloid cells, whereas IRF4 expression has been reported to be lymphoid-restricted. We present evidence that primary murine and human macrophages express IRF4, thereby extending its range of expression to myeloid cells. Here, we provide a comparative analysis of IRF4 and ICSBP expression and function in distinct cell types. These IRF proteins can form specific complexes with the Ets-like protein PU.1, and can activate transcription via binding to PU.1/IRF composite sequences. EMSA analysis revealed that murine macrophages contained both IRF4/PU.1 and ICSBP/PU.1 complexes, analogous to B cells. Over-expression of ICSBP in these macrophages activated transcription of a PU.1/IRF-dependent promoter, whereas over-expression of IRF4 had no effect on this promoter. In contrast, over-expression of either IRF4 or ICSBP in both macrophages and NIH-3T3 fibroblasts suppressed transcription of the PU.1-independent H-2Ld MHC class I promoter. In NIH-3T3 fibroblasts, IRF4 and ICSBP also synergized with exogenous PU.1 to activate transcription of a PU.1/IRF-dependent promoter. Furthermore, both IRF4 and ICSBP activated transcription of the IL-1beta promoter in both cell types. While this promoter is PU.1-dependent, it lacks any known PU.1/IRF composite binding sites. Synergistic activation of the IL-1beta promoter by these IRF proteins and PU.1 was found to require PU.1 serine 148. Together, these data demonstrate that IRF4 and ICSBP are dichotomous regulators of transcription in macrophages.