The biological cost of rifampicin resistance mutations in isogenic isolates of a M.rntuberculosis strain and the development of multidrug resistance in serial clinical Beijingrnisolates were studied. Rifampicin is a major drug used in the treatment of tuberculosis.rnIncreasing rifampicin resistance represents a global clinical problem. Most (about 96%)rnof the resistance to rifampicin is caused by mutations in a small segment of the rpoBrngene, which encodes the p-subunit of RNA polymerase. The effect of three differentrnrpoB mutations on the fitness of M. tuberculosis was examined. Rifampicin-resistantrnmutatnts were initially isolated from a virulent clinical isolate of M. tuberculosis (strainrnHarlingen) at a mutation frequency of 2.3 x 10'8. Mutations in the rpoB gene werernidentified by genotypic sequencing and the growth rates of three defined mutants werernmeasured by competition with the susceptible parent strain in laboratory medium and byrnsingle cultures in a macrophage cell line and in laboratory medium , All mutants exhibitedrna decrease in growth rate compared with the susceptible parent in all three assays. Thernrelative fitness of the mutants varied between 0.29 and 0.96 depending on the mutant andrnassay system used. The results revealed that rifampicin resistance is associated with arncost that is conditional. The Beijing isolates analyzed included eight serial isolates from arnsingle patient in a space of nine years. The RFLP and spoligotype patterns of all thesernisolates were identical. The results from the Beijing isolates showed that Beijingrngenotype is associated an increased tendency toward the development of multidrugrnresistance once resistance to a single first-line drug develops.