Summary. Only in a minority of patients with chronic hepatitis B (CHB) will treatment with interferon (IFN)-α or nucleoside analogues lead to sustained virological response. In vivo immunization (IVI) following virus suppression aims to optimize conditions for an effective immune response: following rapid and profound virus suppression by interferon–lamivudine combination therapy, lamivudine is withdrawn intermittently during continued interferon therapy. It is thought that withdrawal of lamivudine will lead to increased viral replication and increased antigen expression with subsequent immune stimulation. The aim of this prospective pilot study was to evaluate IVI as a therapeutic approach for CHB. Fourteen HBeAg-positive CHB patients were treated for 42 weeks with a combination of pegylated interferon-alpha 2b and lamivudine. After 12 weeks of combination therapy lamivudine was withdrawn intermittently for three consecutive periods of 4 weeks until it was permanently stopped on week 36. At the end of follow-up (week 52) all patients had remained HBeAg positive and the median viral load was similar to baseline. During the initial 12 weeks of treatment, there was a reduction of both the hepatitis B virus (HBV)-specific proliferation capacity of Th-cells and the frequencies of IFNγ-producing cells. During the lamivudine interruption-cycle there was an inverse relation between the increase of HBV-DNA, and the decrease in proliferation capacity and frequency of IFN-γ-producing cells. The intrahepatic fraction of CD8+ T-cells increased during lamivudine withdrawal. In conclusion, IVI was able to transiently stimulate the HBV-specific immune responsiveness of T-cells, but the magnitude of the response was insufficient to cause a beneficial virological effect.