Drug-resistant Mycobacterium tuberculosis is a major threat to public health. In clinical practice, a limited number of resistance mutations in a short sequence of the beta subunit of RNA polymerase (encoded by rpoB) have been described. Spontaneous resistance to rifampin was induced in vitro in M. tuberculosis H37Rv (ATCC 9360). Only three resistance patterns could be detected by PCR-single-strand conformation polymorphism analysis. Sequence analysis revealed that Ser(531)-->Leu arose most frequently, followed by His(526)-->Arg and then either His(526)-->Tyr or His(526)-->Asp. The relative Darwinian fitness of all but one of the mutant genotypes was less than that of the susceptible parent and, for these mutations, there was a significant correlation between fitness and clinical isolation rate (regression analysis P = 0.026). The fitness deficit in some mutants was small, suggesting that there is little likelihood of a spontaneous reversion to susceptibility.