Chromosomal DNA isolated from 3 reference and 2 clinical isolates of Mycobacterium tuberculosis, 2 reference strains each of M. bovis and M. bovis BCG, and 1 reference strain of M. kansasii, was digested with 9 restriction endonucleases. The restriction fragments were analyzed by agarose gel electrophoresis. With each enzyme tested the patterns of the strains of M. tuberculosis, M. bovis, and M. bovis BCG were indistinguishable but clearly distinct from those of M. kansasii. A library of M. tuberculosis H37Rv DNA was prepared by cloning Bam HI digest fragments into lambda 1059. Eight randomly selected clones, having multiple Bam HI sites, were used to probe restriction digests of DNA from the strains of the tuberculosis complex. Two of the cloned segments hybridized with homologous fragments in 4 different enzyme digests of all strains. With 2 clones, hybridization differed among the strains; however, some homologous sequences were detected. With 4 clones, efficient hybridization occurred only with M. tuberculosis H37Rv DNA. These hybridization results indicate that some regions of the chromosome are highly conserved among members of the tuberculosis complex, whereas others are diverged. Selected DNA probes were useful in detecting differences among strains of the tuberculosis complex.