The IS6110 repetitive element is present in multiple copies in most Mycobacterium tuberculosis complex bacteria, except for Mycobacterium bovis strains, which usually contain a single copy of IS6110 located on a 1.9 kb PvuII fragment of the direct repeat region. IS6110 transposition can disrupt coding regions and is a major force of genomic variation. In a previous work it was demonstrated that phospholipase C genes are preferential loci for IS6110 transposition in M. tuberculosis clinical strains. Bacterial phospholipase C enzymes participate in pathogenic mechanisms used by different organisms, and have been implicated in intracellular survival, cytolysis and cell-to-cell spread. Four phospholipase C genes (plcA, plcB, plcC and plcD) were detected in the genomes of M. tuberculosis, Mycobacterium africanum, Mycobacterium microti and 'Mycobacterium canettii'. M. bovis and the vaccine strain M. bovis Bacillus Calmette-Guérin contain only the plcD gene. In the present work, the existence of IS6110 insertions within plcD, the unique phospholipase C gene of M. bovis, has been investigated by PCR, Southern blot hybridization and sequencing analysis. In 18 (7.3 %) of 245 isolates analysed, the plcD gene was interrupted by the insertion of one copy of IS6110, which in all cases was transposed in the same orientation and at the same position, 1 972 894, relative to the genome of M. bovis AF2122/97. These 18 isolates were distributed in 6 different spoligotype patterns and contained 4 to 8 IS6110 copies. In contrast, strains showing an intact plcD gene contained one (87 %), two (9.4 %) or three (2.4 %) IS6110 copies, and only a single isolate (1.2 %) had four IS6110 copies. The implications of plcD gene disruption in M. bovis have not been fully investigated, but no differences in the organ distribution of the disease were detected when animals infected with strains from the same spoligotype patterns bearing plcD : : IS6110 and intact plcD were compared.