Tetanus toxoid (TT) is an antigen known to induce strong T cell specific immune responses in humans after vaccination. Here we have used the Elispot assay to assess the number of TT-specific Interferon-gamma (IFN-gamma) secreting T cells present in individuals and monitored the number of TT specific T cells present in the donors for more than two years. In each of the 22 healthy volunteers tested, TT-specific T cells could be detected. Six out of 7 repetitively tested donors showed a remarkably constant number of TT-specific IFN-gamma secreting T cells over several months, whereas one donor demonstrated a transient increase during a flu-like infection. Three healthy donors received TT booster-immunizations and showed significant increases in the number of TT-specific IFN-gamma secreting T cells which reached peak levels by 4 weeks after vaccination. Depletion of either CD4+ T cells, CD8+ T cells or CD16+/CD56+ T cells by immunomagnetic separation demonstrated that TT-specific IFN-gamma secretion is mediated exclusively by CD4+ T cells. In addition, HLA class-I and -II blocking studies showed that IFN-gamma production is performed by HLA class-II restricted cells. Our data show that the Elispot assay can be reliably used to assess the number of TT-specific CD4+ IFN-gamma producing cells (i.e. probably T helper cells) and therefore maybe also useful for the assessment of reactions to other helper antigens.