Genome deciphering revealed that Mycobacterium tuberculosis encodes a single type II topoisomerase contrary to common bacteria harboring two type II topoisomerases (DNA gyrase and topoisomerase IV). Functions of the M. tuberculosis type II topoisomerase were explored after cloning and expressing the subunits encoding genes in Escherichia coli. M. tuberculosis type II topoisomerase supercoiled relaxed pBR322 with a specific activity close to that of DNA gyrases of common bacteria whereas it exhibited DNA relaxation and formation of cleavable complexes with activities significantly higher than other DNA gyrases. Intermolecular passage activity evaluated by the decatenation of kinetoplast DNA was 25-fold lower than that of the topoisomerase IV from Streptococcus pneumoniae, but was markedly higher than that of the E. coli gyrase. Overall, the type II topoisomerase of M. tuberculosis exhibits classical polyvalent activities of DNA gyrase for supercoiling but enhanced relaxation, cleavage, and decatenation activities.