Chronic hepatitis B virus (HBV) infection is characterized by a weak immune response to HBV. Regulatory T cells (T(reg)) can suppress the function of effector T cells and may thus be key players in this impaired immune response. Changes in the functionality or number of T(reg) could explain the decreased antiviral response in chronic HBV patients. To investigate the role of T(reg) in chronic HBV infection, we compared the proportional frequency and functionality of T(reg) in peripheral blood of 50 chronic HBV patients, 23 healthy controls, and 9 individuals with a resolved HBV infection. A higher percentage of T(reg), defined as CD4, CD25, CD45RO, and cytotoxic T-lymphocyte-associated antigen 4-positive cells, was detected within the population of CD4(+) cells in peripheral blood of chronic HBV patients compared with healthy controls and individuals with a resolved HBV infection. Accordingly, chronic HBV patients displayed a higher FoxP3 messenger RNA level than healthy controls. Depletion of CD25(+) cells from peripheral blood mononuclear cells (PBMC) of chronic HBV patients resulted in an enhanced proliferation after stimulation with HBV core antigen. Reconstitution of these depleted PBMC with CD4(+)CD25(+) T(reg) resulted in a dose-dependent reduction of both HBV-specific proliferation and interferon gamma production. In conclusion, chronic HBV patients harbor an increased percentage of T(reg) in peripheral blood compared with controls. T(reg) have an immunosuppressive effect on HBV-specific T helper cells. The presence of HBV-specific T(reg) could contribute to an inadequate immune response against the virus, leading to chronic infection.