In this paper we describe the development of a host-vector system for genetic studies of fast-growing mycobacteria able to biotransform sterols. A wild strain Mycobacterium smegmatis SN38 and a biotechnological mutant Mycobacterium vaccae B3805 were transformed by electroporation with the pSMT3 E. coli-Mycobacterium shuttle plasmid harbouring the hygromycin resistance gene. Both, the pSMT3 plasmid and its derivative pSMT3-ksdD carrying the 3-ketosteroid-delta 1-dehydrogenase gene (ksdD) from Arthrobacter simplex were stably maintained in M. vaccae B3805. The presence of the pSMT3 vector did not affect biotransformation activities of the host strain. We consider the M. vaccae B3805 strain and the pSMT3 plasmid to be a good host-vector system for cloning in mycobacteria genes coding enzymes involved in steroid degradation pathway.