The ESAT-6 (early secretory antigenic target) molecule is a very important target for T cell recognition during infection with Mycobacterium tuberculosis. Although ESAT-6 contains numerous potential T cell epitopes, the immune response during infection is often focused toward a few immunodominant epitopes. By immunization with individual overlapping synthetic peptides in cationic liposomes (cationic adjuvant formulation, CAF01) we demonstrate that the ESAT-6 molecule contains several subdominant epitopes that are not recognized in H-2(d/b) mice either during tuberculosis infection or after immunization with ESAT-6/CAF01. Immunization with a truncated ESAT-6 molecule (Delta15ESAT-6) that lacks the immunodominant ESAT-6(1-15) epitope refocuses the response to include T cells directed to these subdominant epitopes. After aerosol infection of immunized mice, T cells directed to both dominant (ESAT-6-immunized) and subdominant epitopes (Delta15ESAT-6-immunized) proliferate and are recruited to the lung. The vaccine-promoted response consists mainly of double- (TNF-alpha and IL-2) or triple-positive (IFN-gamma, TNF-alpha, and IL-2) polyfunctional T cells. This polyfunctional quality of the CD4(+) T cell response is maintained unchanged even during the later stages of infection, whereas the naturally occurring infection stimulates a response to the ESAT-6(1-15) epitope that consist almost exclusively of CD4(+) effector T cells. ESAT-6 and Delta15ESAT-6 both give significant protection against aerosol challenge with tuberculosis, but the most efficient protection against pulmonary infection is mediated by the subdominant T cell repertoire primed by Delta15ESAT-6.