Clinical observations suggest that eradication of the hepatitis B virus (HBV) is immune-mediated. Vigorous cytotoxic T lymphocyte (CTL) activity directed at HLA class I–bound viral epitopes are detected during acute hepatitis B, but not in chronic hepatitis B carriers. A CTL epitope derived from the hepatitis B core protein amino acids 18-27 has been incorporated into a vaccine also comprised of a T-helper cell epitope and 2 palmitic acid residues (CY-1899). The aim of this study was to determine whether repeated doses of CY-1899 given to patients with chronic hepatitis B could initiate in vivo CTL activity and viral clearance. Patients with chronic hepatitis B received up to 4 doses (ranging from 0.05 mg to 15 mg) 6 weeks apart. Following vaccination, patients were monitored for hepatitis B surface antigen and “e” status, HBV-DNA levels, liver biochemistry, CTL activity, and any adverse events. Ninety patients with chronic hepatitis B infection received CY-1899. Mean CTL responses were all low but were maximal following vaccination with 5 mg CY-1899. Peak CTL responses never exceeded 10 lytic units (LU) regardless of vaccine dose, this value being well below that seen following resolution of acute hepatitis B. No significant changes in liver biochemistry or viral serology were observed during follow-up. No serious adverse events were noted. Administration of the single-epitope vaccine, CY-1899, initiated CTL activity, but of a magnitude lower than that observed during spontaneous HBV clearance. This low-level CTL activity was not associated with viral clearance.