To improve the current vaccine against tuberculosis, a recombinant strain of Mycobacterium bovis bacillus Calmette-Gue´rin (rBCG) expressing a Mycobacterium tuberculosis vaccine candidate antigen (MPT64) in strong association with the mycobacterial cell wall was developed. To deliver the candidate antigen on the surface, we fused the mpt64 gene to the sequence encoding the PE domain of the PE_PGRS33 protein of M. tuberculosis (to create strain HPE-MPT64-BCG), which we have previously shown to transport proteins to the bacterial surface. In a series of protection experiments in the mouse model of tuberculosis, we showed that (i) immunization of mice with HPE-MPT64-BCG provides levels of protection significantly higher than those afforded by the parental BCG strain, as assessed by bacterial colonization in lungs and spleens and by lung involvement (at both 28 and 70 days postchallenge), (ii) rBCG strains expressing MPT64 provide better protection than the parental BCG strain only when this antigen is surface expressed, and (iii) the HPE-MPT64-BCG-induced MPT64-specific T cell repertoire when characterized by chain variable region- chain joining region (BV-BJ) spectratyping indicates that protection is correlated with the ability to recruit gamma interferon (IFN-)- secreting T cells carrying the BV8.3-BJ1.5 (172 bp) shared rearrangement. These results demonstrate that HPE-MPT64-BCG is one of the most effective new vaccines tested so far in the mouse model of tuberculosis and underscore the impact of antigen cellular localization on the induction of the specific immune response induced by rBCG.