We have developed a gene cloning system for mycobacteria. Based on the nucleotide sequence determined for the M. fortuitum plasmid pAL5000, we have constructed an E. coli/mycobacteria shuttle vector. This vector, pAL8, is composed of pAL5000, pTZ19R (an E. coli plasmid) and a kanamycin resistance gene (from Tn903). We were unable to obtain viable kanamycin resistant pAL8 transformants of M. smegmatis using a PEG-mediated DNA uptake system, in spite of the fact that we could show efficient DNA uptake by transfection using the mycobacterial lytic phage D29. However, kanamycin resistant transformants of M. smegmatis or BCG could be obtained by electroporation. This plasmid cloning system provides a tool for studies of the expression of cloned genes (e.g. virulence) or epitopes in mycobacteria and allows the rational construction of recombinant BCG polyvalent vaccines.