Thermodynamics of 39 oligonucleotides with internal G.T mismatches dissolved in 1 M NaCl were determined from UV absorbance versus temperature profiles. These data were combined with literature values of six sequences to derive parameters for 10 linearly independent trimer and tetramer sequences with G.T mismatches and Watson-Crick base pairs. The G.T mismatch parameters predict DeltaG degrees 37, DeltaH degrees , DeltaS degrees , and TM with average deviations of 5.1%, 7.5%, 8.0%, and 1.4 degrees C, respectively. These predictions are within the limits of what can be expected for a nearest-neighbor model. The data show that the contribution of a single G.T mismatch to helix stability is context dependent and ranges from +1.05 kcal/mol for AGA/TTT to -1.05 kcal/mol for CGC/GTG. Several tests of the applicability of the nearest-neighbor model to G.T mismatches are described. Analysis of imino proton chemical shifts show that structural perturbations from the G.T mismatches are highly localized. One-dimensional NOE difference spectra demonstrate that G.T mismatches form stable hydrogen-bonded wobble pairs in diverse contexts. Refined nearest-neighbor parameters for Watson-Crick base pairs are also presented.