To determine the immunogenicity of two doses of yeast recombinant hepatitis B virus (HBV) vaccine containing surface (S) protein, an open-label, multicenter trial was conducted in 199 healthy HBV-seronegative adults ⩾40 years old. Volunteers were randomly assigned to 1 of 5 groups to receive a total of three 10-µg doses, at 0, 1, and 6 months, or a total of two doses of 20 µg and 10 µg, 20 µg and 20 µg, 40 µg and 10 µg, or 40 µg and 20 µg at 0 and 6 months. The 40-µg/20-µg regimen elicited the highest rate of seroprotection (96.2%), with a geometric mean titer of antibody against the S protein of 369 mIU/mL, not significantly different from the 536 mIU/mL achieved with three doses. These results suggest that a two-dose regimen can achieve seroprotection similar to that of the three-dose regimen. Whether a shorter interval can be used or a booster dose will be needed later to confer durable immunity are unknown.