To further evaluate the human immune response to hepatitis B surface antigen (HBsAg), an enzyme-linked immunosorbent assay [ELISA] was used to measure the in vitro proudction of antibody to HBsAg in normal individuals following booster immunization with the HBsAg vaccine. Following booster immunization, cells spontaneously secreting antibody to HBsAg were present in the peripheral blood. The specific antibody production was first detectable 4 days after booster immunization. The peak of specific antibody production was on day 6, with resolution of the response by day 9. The presence of these spontaneously secreting cells in the peripheral blood precedes the rise in serum antibody levels to HBsAg. The antibody secretion does not require the presence of accessory cells and is not enhanced by pokeweed mitogen, suggesting that these cells are quite advanced in the B-cell maturation sequence. The significance of these observations is discussed. Understanding the human immune response to HBsAg vaccine should provide insights into the immune regulatory abnormalities associated with disease states characterized by chronic hepatitis B antigenemia, including cryoglobulinemia, polyarteritis nodosa and chronic hepatitis.