In recent years, chicken interferon-gamma (ChIFN-gamma) has been identified and cloned from a chicken T cell line. In this study, recombinant ChIFN-gammma produced in the baculovirus and prokaryotic (Escherichia coli) expression systems were characterized and their activity was compared to that of naturally ChIFN-gamma produced by mitogen-activated splenic T cells. The baculovirus-derived ChIFN-gamma protein (Bac-ChIFN-gamma) proved to have physiochemical properties and biological activities similar to those of natural ChIFN-gamma. Indeed, Bac-ChIFN-gamma was able to inhibit the replication of cytolytic viruses in chicken embryo fibroblasts and to activate macrophages, as was determined by nitric oxide production. Levels ranging between 100 and 300 microg/ml of BacChIFN-gamma could be obtained in the supernatants of infected insect cells. On the other hand, yields of the E. coli produced ChIFN-gamma rarely exceeded 100 microg/ml after purification steps and although it was also able to activate the HD11 macrophage cell line in a specific manner, no anti-viral activity could be demonstrated. Therefore, the baculovirus expression system is an appropriate system for the high-level expression of biologically active ChIFN-gamma and will allow further studies of the immunomodulatory and therapeutic effects of this cytokine in vivo.