IFNs are well characterized macrophage-activating agents. Their varied effects are largely mediated via the induction of many genes, whose products act in concert to induce macrophage differentiation. Homologous DNA sequences have been found upstream of the promoter in many of these IFN-inducible genes and bind a family of trans-acting proteins. Interferon consensus sequence binding protein (ICSBP) is one member of this family of interferon regulatory factors (IRF) and is structurally related within the DNA-binding domain to the other members, IRF-1, IRF-2, and ISGF3 gamma. ISCBP mRNA levels become elevated in response to IFN-gamma; however, little is known about the regulation of ICSBP expression at the protein level. In this study, anti-ICSBP peptide Abs were used to quantify and localize ICSBP in murine peritoneal exudate macrophages. Western blot analysis of cytoplasmic and nuclear extracts from treated and control cells revealed ICSBP to be induced by IFN-gamma and not by IFN-alpha and to exist primarily in the nucleus. The regulation of ICSBP induction by IFN-gamma was consistent with the characteristics found at the mRNA level; inhibition by IFN-alpha or glucocorticoids and the requirement for protein kinase C (as determined pharmacologically). The time course of IFN-gamma-induced ICSBP showed an induction of protein that required approximately 12 h to reach maximal levels. Induced ICSBP was relatively stable, exhibiting a half-life of approximately 48 h. Indirect immunofluorescence also demonstrated ICSBP to be an IFN-gamma-inducible protein that is strongly localized to the nucleus.