Streptomycin, the first antibiotic used in tuberculosis control programs, perturbs protein synthesis at the ribosome level. It is shown here that streptomycin resistance in some clinical isolates of Mycobacterium tuberculosis is associated either with missense mutations in the rpsL gene, which encodes ribosomal protein S12, or with base substitutions at position 904 in the 16S rRNA. The primary structure of the S12 protein is well conserved among the mycobacteria, even those, such as M. avium, M. gordonae, and M. szulgai, that are naturally resistant to streptomycin. This suggests that permeability barriers may be responsible for the resistance to the antibiotic.