Tuberculosis is responsible for the deaths of more people each year than any other single infectious disease, with greater than 7 million new cases and 2 million deaths annually. It remains the largest attributable cause of death in HIV-infected individuals, responsible for 32% of deaths of HIV-infected individuals in Africa. The only currently available vaccine for tuberculosis, bacille Calmette-Guerin (BCG) is the most widely used vaccine in the world, being administered to approximately 100 million children each year. Although untoward effects were not seen in several studies of HIV-seropositive children, the safety of live attenuated BCG vaccine in HIV-positive adults remains unknown and a matter of some concern. To obviate potential adverse affects of BCG vaccines in immunodeficient individuals, we have studied five auxotrophic strains of BCG produced by insertional mutagenesis for safety in administration to mice with severe combined immunodeficiency disease (SCID), and for protection in a susceptible strain of mice. The results indicate that viable BCG could no longer be detected in mice receiving the auxotrophs after 16-32 weeks, and that infected SCID mice survived for at least 230 days. In contrast, all SCID mice succumbed within eight weeks to conventional BCG vaccine. When susceptible BALB/c mice were immunized with auxotrophs and subsequently challenged with virulent Mycobacterium tuberculosis, several of the auxotrophs produced comparable protection against intravenous and intratracheal challenge with M. tuberculosis relative to conventional BCG. These results suggest that auxotrophic strains of BCG represent a potentially safe and useful vaccine against tuberculosis for populations at risk for HIV.