The thermodynamics of double-helix formation by (dG-dC)3 in H2O and D2O were measured spectrophotometrically and calorimetrically. Spectrophotometric values based on plots of inverse melting temperature vs. log of concentration are in good agreement with calorimetry. However, spectrophotometric values based on the shape of absorbance vs. temperature curves are in good agreement with calorimetry only if linear pre- and posttransition base lines are subtracted before analysis. The averages of the enthalpies measured by the three most reliable spectroscopic methods are -56.9 and -62.7 kcal/mol of helix in H2O and D2O, respectively. The corresponding calorimetric values are -59.6 and -65.8 kcal/mol of helix.