Southern blotting, sequence analysis and PCR experiments showed that Mycobacterium bovis and Mycobacterium bovis BCG lack a 12.7 kb fragment present in the genome of Mycobacterium tuberculosis. This region is 337 bp downstream of the RD2 region, which was previously described as being absent from some M. bovis BCG strains. The 12.7 kb fragment should be useful as a target for a PCR test to differentiate M. tuberculosis and M. bovis. An analysis of the 12.7 kb region suggests that it represents a deletion in M. bovis rather than an insertion in M. tuberculosis. The deletion removes most of the mce-3 operon, one of four highly related operons which may be involved in cell entry, and therefore it may contribute to differences in virulence or host range in the two species.