Daily parenteral administration of exogenous interferon-γ (IFN-γ) induces or accelerates recovery in experimental and human infections. To develop an alternative delivery system, a replication-defective recombinant adenovirus expressing human IFN-γ was constructed. The complete coding region of IFN-γ was amplified by RT-PCR and inserted into an adenovirus cloning vector under the control of a human cytomegalovirus promoter. Recombinant adenovirus containing the IFN-γ minigene (dAv-IFN-γ) was isolated from 293 cells cotransfected with the linearized plasmid and an El region-deleted fragment of adenovirus genome. Following in vitro infection with dAv-IFN-γ, dose-dependent and time-dependent expression of IFN-γ mRNA and production of soluble protein were demonstrated in human diploid fibroblast and HeLa cell cultures by Northern blot and ELISA, respectively. Extracellular protein secretion persisted for ≥4 weeks following initial transfection, and secreted IFN-γ induced both antiviral activity (8000–25,000 U/ml) and macrophage activation with killing of intracellular Toxoplasma gondii and Leishmania donovani. These results establish that dAv-IFN-γ generates long-term secretion of biologically active IFN-γ in vitro and suggest that this vector may be a useful delivery system for cytokine therapy.