Abstract2‐Deoxystreptamine (2‐DOS) aminoglycosides are a family of structurally related broad‐spectrum antibiotics that are used widely in the treatment of infections caused by aerobic Gram‐negative bacilli. Their antibiotic activities are ascribed to their abilities to bind a highly conserved A site in the 16 S rRNA of the 30 S ribosomal subunit and interfere with protein synthesis. The abilities of the 2‐DOS aminoglycosides to recognize a specific subdomain of a large RNA molecule make these compounds archetypical models for RNA‐targeting drugs. This article presents a series of calorimetric, spectroscopic, osmotic stress, and computational studies designed to evaluate the thermodynamics (ΔG, ΔH, ΔS, ΔCp) of aminoglycoside–rRNA interactions, as well as the hydration changes that accompany these interactions. In conjunction with the current structural database, the results of these studies provide important insights into the molecular forces that dictate and control the rRNA binding affinities and specificities of the aminoglycosides. Significantly, identification of these molecular driving forces [which include binding‐linked drug protonation reactions, polyelectrolyte contributions from counterion release, conformational changes, hydration effects, and molecular interactions (e.g., hydrogen bonds and van der Waals interactions)], as well as the relative magnitudes of their contributions to the binding free energy, could not be achieved by consideration of structural data alone, highlighting the importance of acquiring both thermodynamic and structural information for developing a complete understanding of the drug–RNA binding process. The results presented here begin to establish a database that can be used to predict, over a range of conditions, the relative affinity of a given aminoglycoside or aminoglycoside mimetic for a targeted RNA site vs binding to potential competing secondary sites. This type of predictive capability is essential for establishment of a rational design approach to the development of new RNA‐targeted drugs. © 2003 Wiley Periodicals, Inc. Biopolymers, 2003