We compared cellular immune responses to rectal, subcutaneous, and intradermal administration of Mycobacterium bovis BCG for 5 to 20 weeks in mice, guinea pigs, and macaques. Strong lymphoproliferative responses were induced in spleen cells after in vitro stimulation with purified protein derivative in guinea pigs and macaques, whatever the route of immunization. Comparable high numbers of gamma interferon- and tumor necrosis factor alpha-producing cells were found in the spleen after rectal, subcutaneous, and intradermal immunization of mice and macaques. Similar levels of precursors of cytotoxic T lymphocytes specific for mycobacterial antigens were observed in mice for all immunization routes. In macaques, cytotoxic activity, determined only at the end of the experiment (20 weeks), was similar after rectal and intradermal immunization. Six months after immunization, rectal and subcutaneous routes induced in mice similar levels of protective immunity against challenge with a virulent Mycobacterium tuberculosis strain (H37Rv). Rectal immunization gave immune responses and protective capacity similar to those for parenteral immunization and seemed to be a promising new route of vaccination against tuberculosis; in our study, immunization via the rectal route never induced side effects associated with parenteral routes (axillary adenitis) and could also effectively reduce the risks of viral transmission associated with unsafe injections in the developing world.