BACKGROUND: The frequency of protective antiviral memory B cells after hepatitis B virus (HBV) vaccination is unknown. METHODS: A novel 2-step immunomagnetic protocol to assess the ex vivo frequency of protective HBV surface antigen (HBsAg)-specific memory B cells was used. RESULTS: HBsAg-specific memory B cells were detected in vaccinated individuals, although at very low frequency (median, 0.2% of CD19(+) cells [range, 0%-4% of CD19(+) cells]). No correlation existed between the frequency of HBsAg-specific memory B cells and the corresponding serum antibody titer or B cell enzyme-linked immunosorbent spot findings. CONCLUSION: Our results indicate sustained B cell-mediated protection against HBV despite waning antibody titers, which is consistent with clinical observations.