Resolution of hepatitis B virus (HBV) infection was believed to be attributed to the cytotoxic T cell-mediated killing of infected hepatocytes. However, studies in HBV transgenic mice and HBV-infected chimpanzees revealed that T cell control of HBV replication also involves cytokine-mediated noncytolytic mechanisms. The relative role of cytolytic and noncytolytic functions of virus-specific CD8(+) T cells during interaction with HBV-producing hepatocytes is not well understood. By using HLA-A2 matched effector cells (CD8(+) T cell line or clone) and target cells supporting full HBV replication, we demonstrate that virus-specific CD8(+) T cells can inhibit HBV replication in HBV-producing hepatocytes with minimal cell lysis. Although CD8(+) T cells kill a fraction of infected cells, this effect is minimal, and most of the viral inhibition is mediated by noncytolytic mechanisms. CD8(+) T cells produce an array of cytokines, among which IFN-gamma and TNF-alpha are responsible for HBV inactivation in the target cells. Blockade of IFN-gamma and TNF-alpha abrogated the noncytolytic inhibition of HBV, indicating that these two cytokines mediate the control of HBV by noncytolytic mechanisms. Furthermore, treatment of the HBV-producing hepatocytes with rIFN-gamma and rTNF-alpha resulted in an efficient suppression of viral replication without cytotoxicity. In contrast, coculture of the same target cells with activated HLA-mismatched mitogen-activated lymphomononuclear cells caused a marked cytolytic effect and was less effective in HBV control. These results provide direct evidence that virus-specific CD8(+) T cells efficiently control HBV replication by noncytolytic mechanisms, and this effect is mediated by IFN-gamma and TNF-alpha.