METHODS The insertion sequence IS6110 is widely used as a DNA fingerprinting probe for the classification of Mycobacterium tuberculosis strains. This study has focused on the characterization of regions disrupted by insertion of the IS6110 element. OBJECTIVE To characterize IS6110 insertion loci in clinical isolates of M. tuberculosis, in terms of their genomic location and genetic identity, to ascertain whether IS6110 transposition could be a mechanism driving phenotypic change. METHODS Thirty-three IS6110 insertion loci were cloned from 8 clinical isolates of M. tuberculosis. Clones representing DR locus insertions were identified by hybridization (n = 4), and all other clones were characterized by DNA sequencing (n = 29). The sequence data was analyzed in conjunction with that of 43 other insertion loci identified in published literature and DNA sequence databases. RESULTS The 76 sequences analyzed represented 66 unique insertion loci (including 9 unique insertions into the ipl locus). When mapped to the H37Rv genome, the majority of unique insertion loci demonstrated disruption of coding regions by IS6110 (n = 42; including the ipl insertions), while the remainder either occurred within intergenic regions (n = 17), or could not be mapped to the H37Rv genome sequence (n = 7). Mapping of the insertion loci reveals distribution throughout the chromosome, with isolated preferential insertion loci. CONCLUSIONS This study has demonstrated the occurrence of 66 unique IS6110 insertion loci dispersed throughout the M. tuberculosis genome, with an unexpectedly high incidence of IS6110 insertions occurring within coding regions. However, the IS6110-mediated coding region disruptions identified here may only have limited impact on phenotype, as most of the coding regions disrupted are members of multiple gene families. Disruption of individual members of a family of genes may have no effect on phenotype or could have a minor or major impact, depending on the specificity and activity of the encoded protein.