Chronic hepatitis B is a severe and frequently progressive disease. We assessed the safety and efficacy of thymosin fraction 5 and thymosin-α1 in a prospective, placebo-controlled trial in 12 patients with chronic hepatitis B. All patients had histological and biochemical evidence of active liver disease for at least 6 mo before treatment and were positive for serum hepatitis B virus DNA and HBsAg. Seven patients received thymosin fraction 5 or thymosin-α1 and five patients received placebo twice weekly for 6 mo. By the conclusion of the study (1 yr), serum aminotransferase levels had improved significantly in thymosin-treated patients, but not in the placebo group. Six (86%) of the thymosin treated patients and one (20%) patient given placebo cleared hepatitis B virus DNA from serum (p < 0.04, Fisher's exact test). After treatment, replicative forms of hepatitis B virus DNA were present in the liver specimens of four of five placebo-treated patients but in only one of seven thymosin-treated patients (p < 0.04, Fisher's exact test). Response to thymosin therapy was associated with significant improvements in peripheral blood lymphocyte and CD3 and CD4 counts and in in vitro production of interferon-γ over initial values. No significant side effects were observed in patients given thymosin or in placebo-treated patients. Clinical, biochemical and serological improvement in patients responding to thymosin were sustained during 26 ± 3 mo of follow-up. The results of this pilot trial suggest that thymosin therapy promotes disease remission and cessation of hepatitis B virus replication in patients with chronic viral infection.