Supernatants of the chicken T cell line 855 contain antiviral and macrophage-activating factor activity and strongly activate transcription of the guanylate-binding protein (GBP) gene in chicken cells. To characterize the cytokine responsible for the GBP-inducing activity, we chose a cDNA expression cloning strategy in COS cells. Sequencing a positive clone revealed that it encodes chicken interferon-γ (ChIFN-γ). Histidine-tagged ChIFN-γ was expressed in Escherichia coli and purified by nickel chelate affinity chromatography. ChIFN-γ from COS cells and E. coli both potently induced GBP RNA synthesis but were rather poor antiviral agents. In macrophages, recombinant ChIFN-γ strongly stimulated secretion of nitric oxide and enhanced expression of major histocompatibility complex class II antigen. A rabbit antiserum to E. coli-derived ChIFN-γ effectively neutralized the macrophage-activating factor activity secreted by concanavalin A-induced spleen cells and various T cell lines, suggesting that IFN-γ is the major macrophage-activating factor of the chicken.