Aminoglycosides have been at the forefront of antimicrobial therapy for the past 50 years. Their specificity is believed to lie in binding duplex RNAs (rRNA). Competition dialysis studies of various nucleic acid forms with 9-aminoacridine, quinacrine, and a neomycin-acridine conjugate were carried out. Our results suggest a strong preference for aminoglycoside binding to nucleic acids that can adopt an A-type conformation. These results challenge the common belief that aminoglycoside specificity is simply for duplex RNAs.