High-efficiency transformation of DNA is integral to the study of mycobacteria, allowing genetic manipulation. Electroporation is the most widely used method for introducing DNA into mycobacterial strains. Many parameters contribute to high-efficiency transformation; these include the species per strain, the transforming DNA, the selectable marker, the growth medium additives, and the conditions of electroporation. In this chapter we provide an optimized method for the transformation of representative slow- and fast-growing species of mycobacteria-Mycobacterium tuberculosis and M. smegmatis, respectively.