Widespread use of DNA restriction fragment length polymorphism (RFLP) to differentiate strains of Mycobacterium tuberculosis to monitor the transmission of tuberculosis has been hampered by the need to culture this slow-growing organism and by the level of technical sophistication needed for RFLP typing. We have developed a simple method which allows simultaneous detection and typing of M. tuberculosis in clinical specimens and reduces the time between suspicion of the disease and typing from 1 or several months to 1 or 3 days. The method is based on polymorphism of the chromosomal DR locus, which contains a variable number of short direct repeats interspersed with nonrepetitive spacers. The method is referred to as spacer oligotyping or "spoligotyping" because it is based on strain-dependent hybridization patterns of in vitro-amplified DNA with multiple spacer oligonucleotides. Most of the clinical isolates tested showed unique hybridization patterns, whereas outbreak strains shared the same spoligotype. The types obtained from direct examination of clinical samples were identical to those obtained by using DNA from cultured M. tuberculosis. This novel preliminary study shows that the novel method may be a useful tool for rapid disclosure of linked outbreak cases in a community, in hospitals, or in other institutions and for monitoring of transmission of multidrug-resistant M. tuberculosis. Unexpectedly, spoligotyping was found to differentiate M. bovis from M. tuberculosis, a distinction which is often difficult to make by traditional methods.