Mycobacterium smegmatis inactivates rifampin by ribosylating this antibiotic. The gene responsible for this ability was cloned and was shown to confer low-level resistance to this antibiotic (MIC increase, about 12-fold) in related organisms. A 600-bp subclone responsible for ribosylating activity and resistance carried an open reading frame of 429 bp. Targeted disruption of the gene in M. smegmatis resulted in mutants with much increased susceptibility to rifampin (MICs of 1.5 instead of 20 microg/ml) as well as the loss of antibiotic-inactivating ability. Also, disruption of this gene led to a much lower frequency of occurrence of spontaneous high-level rifampin-resistant mutants.