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 encode chicken interferon-gamma (ChIFN-gamma). Histidine-tagged ChIFN-gamma was expressed in Escherichia coli and purified by nickel chelate affinity chromatography. ChIFN-gamma from COS cells and E. coli both potently induced GBP RNA synthesis but were rather poor antiviral agents. In macrophages, recombinant ChIFN-strongly stimulated secretion of nitric oxide and enhanced expression of major histocompatibility complex class II antigen. A rabbit antiserum to E. coli derived ChIFN-gamma effectively neutralized the macrophage-activating factor activity secreted by concanavalin A-induced spleen cells and various T cell lines, suggesting that IFN-gamma is the major macrophage-activating factor of the chicken.