Site-specific integration into the mycobacterial chromosome can produce stable transformants useful for understanding pathogenesis. However, gene expression can be problematic at certain sites of integration. We have used the Streptomyces C31 integration system to integrate vector DNA into Mycobacterium smegmatis, M. bovis BCG, and M. tuberculosis through site-specific recombination. A single dominant insertion site was found in M. smegmatis, as previously reported. Three different insertion sites were found in M. bovis BCG. In M. smegmatis, integrated vectors appear to be far more stable than episomal plasmids during unselected passage in vitro, although excision products are detectable. Plasmids based on the C31 integration system could make useful tools for the study of mycobacterial genetics.