The primary activation of T-helper and T-cytotoxic cells following mucosal immunization with recombinant Streptococcus gordonii was studied in vivo by adoptive transfer of ovalbumin (OVA)-specific transgenic CD8(+) (OT-I) and CD4(+) (OT-II) T cells. A recombinant strain, expressing on the surface the vaccine antigen Ag85B-ESAT-6 from Mycobacterium tuberculosis fused to OVA T-helper and T-cytotoxic epitopes (peptides 323 to 339 and 257 to 264), was constructed and used to immunize C57BL/6 mice by the intranasal route. Recombinant, but not wild-type, bacteria induced OVA-specific CD4(+) and CD8(+) T-cell clonal expansion in cervical lymph nodes, lung, and spleen. OVA-specific CD4(+) and CD8(+) T-cell proliferation appeared first in cervical lymph nodes and later in the spleen, suggesting a possible migration of activated cells from the inductive site to the systemic district. A significant correlation between the percentages of CD4(+) and CD8(+) proliferating T cells was observed for each animal. The expression of CD69, CD44, and CD45RB on proliferating T lymphocytes changed as a function of the cell division number, confirming T-cell activation following the antigen encounter. These data indicate that intranasal immunization with recombinant S. gordonii is capable of inducing primary activation of naive antigen-specific CD4(+) and CD8(+) T cells, both locally and systemically.