Mycobacterium tuberculosis isolates from 167 patients attending three London hospitals were analyzed by two techniques for strain differentiation. A significant number of isolates that appeared identical with the recently developed spoligotyping system could be distinguished from each other by IS6110 restriction fragment length polymorphism analysis, with the latter technique demonstrating a generally higher level of discrimination. Spoligotyping, on the other hand, was particularly useful for analysis of isolates with low IS6110 copy numbers, and use of the two techniques in tandem provided an optimal approach to the identification of clusters with epidemiological evidence consistent with recent transmission. Spoligotyping can be applied directly to clinical samples by PCR and provides an important tool for the rapid detection of nosocomial transmission of individual strains.