The effects of tumor necrosis factor-a and/or interferon-γ on the replication of hepatitis B virus were examined using HB611 cells. These cells were derived from human hepatoblastoma cells, Huh6, by integrating hepatitis B virus DNA, and produce hepatitis B virus continuously. Each of the cytokines inhibited hepatitis B virus replication in the cells assessed as the amount of episomal hepatitis B virus DNA, without a decrease in cell viability. When the two cytokines were administered together, the inhibitory effect became greater. Incubation of the cells with 1, 000 U/ml tumor necrosis factor-α decreased HBV DNA replicative intermediates by 55%, and that with 1, 000 U/ml interferon-γ decreased these by 51%. Furthermore, incubation with 1, 000 U/ml tumor necrosis factor-α and 1, 000 U/ml interferon-γ in combination decreased HBV DNA replicative intermediates by 71%. In contrast, the amount of hepatitis B virus RNA and secretion of hepatitis B e antigen were not apparently reduced by the cytokines, and 2′, 5′-oligoadenylate synthetase activity was not detected in the supernatant. These results suggest that tumor necrosis factor-α and interferon-γ inhibit hepatitis B virus replication by blocking some step in reverse transcription and that the 2′, 5′-oligoadenylate synthetase is not involved in the mechanism underlying the inhibition by these two cytokines. © Wiley-Liss, Inc.