We have investigated the vaccine potential of two peptides derived from the 6-kDa early secretory antigenic target (ESAT)-6 antigen in the mouse model of tuberculosis. The peptides were both strongly immunogenic in B6CBAF1 (H-2b,k) mice and primed recall responses of the same intensity after immunization. However, both tuberculosis infection and immunization with ESAT-6 resulted in responses focused towards ESAT-61-20. Multiple antigen peptide constructs as well as free peptides were emulsified with dimethyl dioctadecylammonium bromide/monophosphoryl lipid A/IL-2 and tested as experimental vaccines in an i.v. and aerosol model of tuberculosis in mice. The peptide were highly immunogenic and induced cellular responses of the same magnitude. However, only vaccines based on the subdominant ESAT-651-70 epitope promoted significant levels of protective immunity and the level of protection was equivalent to that achieved with ESAT-6 and BCG. These findings demonstrate the potential of peptide-based vaccines against tuberculosis and indicate that there is not direct correlation between the hierarchy of response to naturally processed peptides and their ability to induce protective immunity against Mycobacterium tuberculosis.