DNAs from nine mycobacterial species were cleaved with different restriction endonucleases and hybridized with cDNA probes synthesized to total transfer RNAs (tRNAs) from Mycobacterium smegmatis and M. tuberculosis. The hybridization data indicate that tRNA genes are conserved but their gross genomic organization has diverged in six of the nine species examined. Species of the MTB complex appeared to have identical tRNA gene organization. Hybridization with cDNAs synthesized to 23S plus 16S rRNAs from Escherichia coli indicate that the tRNA genes map near the rRNA genes. Recombinant plasmids, pSB1, pSB2, pSB4 and pSB8 encoding tRNA(s) and rRNA(s) were isolated from a gene bank of M. tuberculosis H37Rv. Using pSB2 as probe, a SalI RFLP was observed that distinguishes the virulent and avirulent strains of M. tuberculosis H37Rv and H37Ra, respectively.