Site-specific recombinases such as the Saccharomyces cerevisiae Flp and the P1 phage Cre proteins have been increasingly used for the construction of unmarked deletions in bacteria. Both systems consist of an antibiotic resistance gene flanked by recognition sites in direct orientation and a curable plasmid for temporary expression of the respective recombinase gene. In this chapter, we describe strategies and methods of how to use sequence-specific recombination mediated by Flp and Cre to construct mutants of Mycobacterium smegmatis , Mycobacterium bovis BCG, and Mycobacterium tuberculosis .