A small (40 nucleotides) stem-loop derivative (J6f1) of a specific aminoglycoside-binding RNA aptamer, containing a 3 nt and a 1 nt bulge, has previously been shown to stoichiometrically bind tobramycin with a dissociation constant of approximately 5 nM [Hamasaki, K., Killian, J., Cho, J. and Rando, R. R. (1997) Biochemistry 36, 1367-1371]. This construct can strongly discriminate among similar aminoglycosides with respect to binding. A combination of chemical interference studies, chemical modification studies, and mutational studies are performed to define the aminoglycoside binding site of J6f1. Recognition of the aminoglycoside by J6f1 involves contacts with nucleotide bases, rather than with the phosphate backbone. The binding site 1 comprised of part of the stem-loop region. The two bulges are also essential for high affinity and stoichiometric binding of tobramycin. These bulges are probably important for prying open the double helical region, thereby allowing the aminoglycoside access to the nucleotide bases.