BACKGROUND AND AIM: Thymosin-alpha1 (T-alpha1) influences T-cell maturation, production of Th1-type cytokines, and activity of NK cell-mediated cytotoxicity. The aim of this study is to evaluate the types of lymphocytes that contribute to the reduction in viral load in patients with chronic hepatitis B (CHB) following T-alpha1 treatment. METHODS: Seven patients with CHB were treated with 1.2 mg of T-alpha1 for 24 weeks. Peripheral blood lymphocytes (PBL) and intrahepatic lymphocytes were analyzed by flow cytometry. Serum cytokines (IL-4 and IFN-gamma) were measured by ELISA. RESULTS: Forty-eight weeks after T-alpha1 treatment, two patients (28.6%) showed normalized alanine aminotransferase and decreased HBV-DNA to undetectable level from serum. The histology activity index score significantly decreased (P<0.05). Although elevated serum interferon (IFN)-gamma was not observed, IFN-gamma producing Th1-type CD4(+) PBL appeared to be increased. CD56(+) natural killer T (NKT) cells in PBL did not increase, these cells in the liver remained significantly augmented even at the end of treatment (P<0.05). CD57(+) NKT cells slightly increased and the ratio of CD4(+) T/CD8(+) T cells decreased in the liver. T-alpha1 did not influence either double-positive CD4(+)8(+) T or double-negative CD4(-)8(-) cell subsets. CONCLUSION: NKT cells and CD8(+) cytotoxic T lymphocytes augmented in the liver by T-alpha1 may eliminate hepatitis B virus infected hepatocytes.