Gene-inactivation techniques were employed to construct a eubacterial organism harbouring a single functional rRNA operon. This mutant of Mycobacterium smegmatis permits replacement of the single remaining rRNA operon with a homologous fragment from a vector-borne gene. By homologous recombination with the chromosome a plasmid-borne rDNA segment with resistance markers substitutes for the corresponding region of the chromosomal rRNA operon, resulting in a homogeneous population of mutated ribosomes in the cell. As a first result we demonstrate that the single allelic knock-out strain allows for isolation of rRNA mutants with a drug-resistant phenotype, circumventing the problem of recessivity which prohibits the isolation of such mutants in organisms with multiple rRNA operons. Subsequently, by allelic exchange experiments, it was demonstrated that the rRNA mutation found indeed confers drug resistance in vivo. This system provides intriguing potential for the study of the structure and function of ribosomal RNAs.