Immunization by intramuscular injection of plasmid DNA expressing mycobacterial 65-kDa heat shock protein (hsp65) protects mice against challenge with virulent Mycobacterium tuberculosis H37Rv. During infection or after immunization, CD4⁺/CD8⁻ and CD8⁺/CD4⁻ hsp65-reactive T cells increased equally in spleens. During infection, the majority of these cells were weakly CD44 positive (CD44^lo) and produced interleukin 4 (IL-4) whereas after immunization the majority were highly CD44 positive (CD44^hi) and produced gamma interferon (IFN-γ). In adoptive transfer of protection to naive mice, the total CD8⁺/CD4⁻ cell population purified from spleens of immunized mice was more protective than that from infected mice. When the cells were separated into CD4⁺/CD8⁻ and CD8⁺/CD4⁻ types and then into CD44^hi and CD44^lo types, CD44^locells were essentially unable to transfer protection, the most protective CD44^hi cells were CD8⁺/CD4⁻, and those from immunized mice were much more protective than those from infected mice. Thus, whereas the CD44^lo IL-4-producing phenotype prevailed during infection, protection was associated with the CD8⁺/CD44^hiIFN-γ-producing phenotype that predominated after immunization. This conclusion was confirmed and extended by analysis of 16 hsp65-reactive T-cell clones from infected mice and 16 from immunized mice; the most protective clones, in addition, displayed antigen-specific cytotoxicity.