Convincing correlates of protective immunity against tuberculosis have been elusive. In BALB/c mice, intra-nasal immunization with a replication-deficient recombinant adenovirus expressing Mycobacterium tuberculosis antigen 85A induces protective lower respiratory tract immunity against pulmonary challenge with Mycobacteriun tuberculosis, while intra-dermal immunization with adenovirus-85A does not. Here we report that intra-nasal immunization with adenovirus-85A induces expression of the chemokine receptor CXCR6 on lung CD8 T lymphocytes, maintained for at least 3 months. CXCR6 positive antigen-specific T cell numbers are increased among broncho-alveolar lavage-recoverable cells. Similarly, intra-nasal immunization with recombinant antigen 85A with adjuvant induces CXCR6 expression on lung CD4 cells in BALB/c and C57Bl/6 mice, while a synthetic ESAT6(1-20) peptide with adjuvant induces CXCR6 expression in C57Bl/6 mice. Parenteral immunization fails to do so. Up-regulation of CXCR6 is accompanied by a transient elevation of serum CXLC16 after intra-nasal immunization and lung cells cultured ex vivo from mice immunized intra-nasally show increased production of CXCL16. Administration of CXCL16 and cognate antigen intra-nasally to mice previously immunized parenterally increases the numbers of antigen-specific T lymphocytes in the broncho-alveolar lavage-recoverable population, which mediate inhibition of the early growth of Mycobacterium tuberculosis after challenge. We conclude that expression of CXCR6 on lung T lymphocytes is a correlate of local protective immunity against Mycobacterium tuberculosis after intra-nasal immunization and that CXCR6 and CXCL16 play an important role in the localization of T cells within lung tissue and the broncho-alveolar lavage-recoverable compartment.