RNA constructs prepared from wild-type and mutant (1555(G)) human mitochondrial 12S RNA were studied with respect to their abilities to specifically bind aminoglycoside antibiotics. The 1555(G) point mutation had previously been found to be associated with hereditary deafness induced by aminoglycosides. It is shown here that the 1555(G) RNA analog stoichiometrically binds aminoglycosides with high affinities, while the wild-type construct does not bind aminoglycosides at all. Analogous mutations in a 16S bacterial rRNA construct show the opposite behavior. Bacterial 16S rRNA constitutes the functional target for aminoglycoside antibiotics. While the wild-type 16S rRNA decoding region construct binds aminoglycosides stoichiometrically with binding affinities in the micromolar range, the mutant is unable to specifically bind aminoglycosides. These results demonstrate the importance of a specific GC base pair in aminoglycoside binding in both the human and the bacterial rRNA constructs. These studies also provide quantitative evidence in support of the hypothesis that the 1555(G) point mutation in human mitochondrial 12S RNA causes aminoglycoside induced deafness.