Gene replacement is a powerful technique that exploits the innate ability of an organism to recombme homologous regions of DNA. It allows for the specrfic replacement of targeted genomic sequences with copies of those sequences carrying defined mutations, and therefore can facilitate the assignment of function to cloned genes. Clearly there are many potential uses for gene-replacement methods in mycobacterial research. Exploitatton of the vast amount of information coming from the Mycobacterium tuberculosis genomesequencing proJect will, in many cases, require the abihty to confirm efficiently gene assignments based on sequence homologies. The desirability of ratronally developing alternatives to the currentMycobacterium bovis Bacille Calmette-Guerm (BCG) vaccme also requires that this technique be extended to allow the use of selective markers for gene-replacement events, which are acceptable for use in vaccines to be administered to the human population