The presence of two insertion sequences, IS406 and IS407, was tested by polymerase chain reaction (PCR) amplification in 25 strains representing 15 Burkholderia species and the close relative Ralstonia pickettii. A total of 50% of the 25 strains contained at least one of the two insertion sequences (ISs) and a statistically significant correlation was found between the occurrences of IS406 and IS407. Moreover, PCR-RFLP studies of the amplified fragments showed that IS406 is largely conserved among all the strains tested, whereas IS407 is rather polymorphic. Transposition activity was studied in Burkholderia vietnamiensis TVV75, using the pGBG1 target plasmid. This entrapping plasmid permitted the isolation and characterization of three active IS, able to activate the plasmid-borne tetA gene after transposition. Sequencing permitted the identification of these mobile genetic elements as isoforms of IS402, IS407 and IS1416. PCR amplification products provided IS probes, which were used to determine the copy-numbers of IS402, IS407 and IS1416 in the genome of B. vietnamiensis TVV75, by Southern blotting. Copy numbers are 12, 3 and 11 respectively. To our knowledge, this is the first description of active insertion sequences in B. vietnamiensis.