Long-term protection after hepatitis B vaccination is dependent on the persistence of a strong immunologic memory. In search of reliable markers for a hepatitis B surface antigen (HBsAg)-specific immunological memory we studied the cellular and humoral immune responses of 15 healthy individuals who were successfully vaccinated but had lost anti-HBs titers. To determine the reactivity of vaccine-induced HBsAg-specific T cells of both effector and memory phenotype CD4+/CD45RA+ and CD4+/CD45R0+ T cells, respectively, were isolated, stimulated with HBsAg and tested for IFN-gamma and IL-5-secretion by enzyme-linked immunospot assays (Elispot). To detect even small numbers of specific T cells, we enriched the appropriate subpopulation from the entire PBMC population. B cell memory was analysed by cocultivation of isolated B cells with CD4+ T cells and identification of anti-HBs-secreting cells by Elispot. All individuals were revaccinated and humoral and cellular responses were determined. The results showed significant numbers of HBsAg-specific memory T and B cells present in all vaccinees despite the absence of specific antibodies. Our data suggest that individuals who had lost their anti-HBs seropositivity still show immunologic T cell memory and that these T cells are able to trigger anti-HBs production of B cells activated by revaccination.