Two methods: triparental conjugation and electrotransformation were used for introduction of plasmid DNA into mycobacterial cells. The introduction of shuttle plasmid pMY10 into M. fortuitum mutant caused the activation of its chromosomal cryptic KmR gene. The used of integration vector pUS 903 allowed to obtain a collection of mutants interesting for studies of genetic determination of steroid biotransformation and drug-resistance in mycobacteria.