Mycobacteria are major pathogens of man and animals. There are approximately 10 million cases of tuberculosis world wide with an annual mortality of three million people. Leprosy, caused by Mycobacterium leprae, afflicts over ten million people, primarily in developing countries. M. tuberculosis and mycobacteria of the M. avium-intracellulare-scrofulaceum (MAIS) group are major opportunistic pathogens of patients with acquired immune deficiency syndrome (AIDS). M. paratuberculosis is the cause of Jöhne's disease in cattle. Yet, BCG (Bacille Calmette-Guerin), an avirulent strain of M. bovis, is the most widely used human vaccine in the world, having been administered to about 2.5 X 10(9) people since 1948 (ref. 4). BCG was highly protective against tuberculosis in England, but has been found not to be effective in preventing pulmonary tuberculosis in adults in Southern India. We have initiated studies to develop the methodology for efficient gene transfer in mycobacteria. We have constructed recombinant shuttle phasmids which are chimaeras containing mycobacteriophage DNA into which an E. coli cosmid is inserted. They can replicate in E. coli as plasmids and in mycobacteria as phages, and transfer DNA across both genera. These shuttle vectors permit for the first time the introduction of foreign DNA by infection into M. smegmatis and BCG. By introducing and ultimately expressing genes for protective antigens for a variety of pathogens, it may be possible to develop cultivatable mycobacteria into useful multivaccine vehicles.