The recent emergence of indolent and rapidly fatal drug-resistant strains of Mycobacterium tuberculosis has renewed interest in defining the molecular mechanisms of drug resistance in the tubercle bacilli. In this report, we have examined the mechanism of resistance to streptomycin (Sm) in M. tuberculosis through the cloning and nucleotide sequence analysis of the gene encoding the ribosomal S12 protein (rpsL gene) from streptomycin-resistant strains and their streptomycin-sensitive parental strains. We have demonstrated that five singly SmR M. tuberculosis strains and an SmR isolate that has reduced sensitivity to multiple antibiotics have identical point mutations at codon 43 of the rpsL gene. Mutations at this same site confer SmR in Escherichia coli. In contrast, two other multiple drug-resistant M. tuberculosis strains that are resistant to Sm have rpsL genes that have the same nucleotide sequence as their drug-sensitive parent strains, suggesting that different resistance mechanisms are involved in these strains.