The specificity of neomycin B and related aminoglycoside antibiotics in their interaction with Rev responsive element (RRE) of HIV-1 mRNA has been studied directly observing the aminoglycoside-RNA complexes using surface plasmon resonance. Several different RNA sequences, each with a biotin tag, have been prepared using T7 RNA polymerase-catalyzed transcription of synthetic DNA templates and have been immobilized on a streptavidin-coated surface for the binding study. The results indicuate the neomycin B is not specific for the G-rich bubble region in RRE. Rather, it appears to interact with three different sites, each with a submicromolar dissociation constant, wihtin the 67-nucleotide domain II of RRE. Further analysis of neomycin B binding with three short synthetic RNA hairpins showed binding with submicromolar affinity and 1:1 stiochiometry in each case. This suggest that neomycin B may generally bind with this affinity to regular AS-form RNA or hairpin loops. The approach described here is generally useful for understanding the fundamental interactions involved in the specific recognition of nucleic acids by small molecules which is the basis of rational drug design.