Thymalfasin (thymosin alpha-1; Talpha1) is a 28-amino acid polypeptide that has shown efficacy in the treatment of chronic hepatitis B virus (HBV) infection. The objective of this study was to evaluate the long-term, dose-related efficacy and safety of Talpha1 treatment in chronic hepatitis B patients with positive HBV-DNA and abnormally high alanine aminotransferase (ALT) levels. A total of 316 patients were randomized to receive either 0.8 or 1.6 mg of Talpha1 monotherapy for 24 weeks. At the end of the 72-week observation period (12 months after cessation of therapy), 36.4% of patients in the 1.6-mg treatment group achieved normalization of ALT, 30% achieved clearance of HBV-DNA by branched DNA vs 15% by transcription-mediated amplification, and 22.8% achieved clearance of HBe-antigen. Patients in the 0.8-mg treatment group achieved similar efficacy rates, although patients with advanced fibrosis demonstrated a significantly better response rate when treated with 1.6 mg of Talpha1 monotherapy vs 0.8 mg (as determined by intragroup analysis; patients were not stratified by liver biopsy). All adverse drug reactions were mild and most involved the fluctuation of liver enzymes, which was most likely related to the positive immune effects caused by the response to Talpha1 treatment. Adverse event incidence was similar in the 1.6- and 0.8-mg treatment groups. In conclusion, Talpha1 at doses of 0.8 and 1.6 mg exhibits long-term efficacy against hepatitis B with a good safety profile.