The transient nature of adenovirus-mediated transgene expression has been attributed to adaptive immune responses to adenoviral proteins and transgene products. However, the cytokines interferon-gamma (IFNgamma) and tumor necrosis factor-alpha (TNFalpha) inhibit transgene expression from adenoviral vectors in vitro by a transcription-related mechanism, and their early induction following vector administration in vivo suggests a contribution of innate immunity in regulating transgene expression. In this study, the significance of cytokine expression and its relation to adaptive and innate immunities were determined in TNFalpha-knockout mice, IFNgamma-knockout mice, or anti-IFNgamma mAb-injected animals. Adenoviral LacZ reporter expression directed by human cytomegalovirus (HCMV) promoters was greater in magnitude and duration than that by the murine CMV (MCMV) promoter. beta-Galactosidase reporter gene expression up to day 7 was greater in cytokine-deficient animals compared with wild type. Decrements in transgene expression occurred in advance of adaptive immune responses and were not due to alterations in specific adaptive immunity or vector clearance in cytokine-depleted mice. We conclude that TNFalpha and IFNgamma inhibit early adenovirus-mediated transgene expression by HCMV and MCMV promoters in vivo. Cytokine inhibition of expression is independent of adaptive immunity and is likely secondary to innate immune responses to adenovirus infection.