A method for the isolation of DNA from mycobacteria propagated in vitro is described that utilizes organic solvents to extract lipoidal components from the outer membrane, and digestion with a protease (nagarse) and lysozyme to penetrate the cell wall. The mycobacterial cells were lysed by the addition of detergent and the DNA was purified by digestion with pronase, sequential phenol and chloroform extractions, and digestion with RNAase A. The isolated DNA, which was obtained in good yields, was of a relatively high Mr and could be readily digested by restriction endonucleases. By this method, the genomes of Mycobacterium avium, M. intracellulare, M. lepraemurium, 'M. lufu', M. marinum, M. phlei, M. scrofulaceum, M. smegmatis and M. tuberculosis were isolated and the restriction endonuclease digestion patterns analysed. Each species could be distinguished by the digestion patterns, indicating that this approach can be used for identifying mycobacterial species. This approach is also sufficiently sensitive to differentiate strains since we were able to distinguish two independently isolated strains of M. tuberculosis, H37 and H4. In addition, no evidence was obtained for the presence of methylcytosine residues in the sequences 5'.CCGG.3',5'.CCCGGG.3',5'.CC(A/T) GG.3' or for methyladenine at 5'.GATC.3' in the DNA of the nine mycobacterial species examined using pairs of restriction enzymes that recognize and cleave at the same nucleotide sequence but differ in their sensitivity to 5-methylcytosine or 6N-methyladenine.