Isothermal titration calorimetry (ITC), computational, and osmotic stress techniques have been used to characterize the changes in heat capacity, solvent-accessible surface, and hydration that accompany the binding of the aminoglycoside paromomycin to both prokaryotic and eukaryotic rRNA A-site model oligonucleotides. Regarded as a whole, the results of these studies suggest that the intrinsic heat capacity change (DeltaC(p)) for the binding of paromomycin to each rRNA A-site is near zero, with the negative DeltaC(p) observed for the binding of the drug to the prokaryotic rRNA A-site being dictated by the coupled destacking of the adenine residues at positions 1492 and 1493. In this connection, DeltaC(p) provides a useful calorimetric signature for assessing the relative impacts of novel and existing A-site targeting ligands on rRNA conformation, which, in turn, should provide a useful analytical tool for facilitating the drug design process, since aminoglycoside-induced destacking of All 492 and A1493 is thought to be a determining factor in the mistranslational and antimicrobial activities of the drugs.