The 19-kD antigen is a cell wall-associated lipoprotein present in Mycobacterium tuberculosis and in bacille Calmette-Guérin (BCG) vaccine strains. Expression of the 19-kD antigen as a recombinant protein in two saprophytic mycobacteria-M. vaccae and M. smegmatis-resulted in abrogation of their ability to confer protection against M. tuberculosis in a murine challenge model, and in their ability to prime a DTH response to cross-reactive mycobacterial antigens. Induction of an immune response to the 19-kD antigen by an alternative approach of DNA vaccination had no effect on subsequent M. tuberculosis challenge. These results are consistent with a model in which the presence of the 19-kD protein has a detrimental effect on the efficacy of vaccination with live mycobacteria. Targeted inactivation of genes encoding selected antigens represents a potential route towards development of improved vaccine candidates.