The thermal unfolding of two RNA hairpin systems derived from the aminoacyl accepting arm of Escherichia coli tRNA(Ala) that included all possible single internal mismatches mostly in the third base pair position was measured spectroscopically in 0.1 M NaCl at pH 7.5 and, in part, 5.5. The thermodynamic parameters DeltaH(o), DeltaS(o), DeltaG(o), and T(m) of a total of 36 RNA strands were determined through nonlinear curve fitting of the melting profiles (22 tetralooped 22mers and 14 heptalooped 25mers, same stem sequence). Only three of the 22mers, the A.C-containing variants, were shown to be significantly more stable at pH 5.5. A number of remarkable differences-most likely of more general relevance-between the thermodynamics of certain structurally very similar hairpin variants (e.g., G.C versus A.U, G.U versus I.U) at pH 7.5 are discussed with respect to two possible ways of helix stabilization: pronounced hydration versus low entropic penalty. Four selected 22mers were additionally analyzed in 1 M NaCl and in solvent mixtures containing ethanol, ethylene glycol, and dimethylformamide. The wealth of thermodynamic data suggest that the exothermicity DeltaH(o) and entropic penalty T x DeltaS(o) of folding are strongly dominated by the rearrangement and formation of hydration layers around the solutes, while it is well-known that the stability of folding results only from the difference (DeltaG(o)) and ratio of both parameters (T(m) = DeltaH (o)/DeltaS(o)).