The memory/activated T cells, which mediate the long-lived host response against tuberculosis, in mice immunized with either bacillus Calmette-Guérin (BCG) or mycobacterium heat-shock protein 65 (hsp 65) antigen expressed from plasmid DNA (DNA-hsp 65), were characterized. Protection against Mycobacterium tuberculosis challenge by DNA-hsp 65 vaccination was associated with the presence of lymph node T-cell populations in which CD8+/CD44hi interferon-gamma (IFN-gamma)-producing/cytotoxic cells were prominent even after 8 or 15 months of plasmid DNA-mediated immunizations, whereas after BCG vaccination the majority were CD4+/CD44lo IFN-gamma-producing T cells. When the cells were separated into CD4+CD8- and CD8+CD4- and then into CD44hi and CD44lo types, CD44lo cells were essentially unable to transfer protection in adoptive transfer experiments, the most protective CD44hi cells were CD8+CD4- and those from DNA-vaccinated mice were much more protective than those from BCG-immunized mice. The frequency of protective T cells and the level of protection were increased up to 8 months and decreased after 15 months following DNA or BCG immunizations.