The thermodynamics of the B to Z transition in poly(dGdC) was examined by differential scanning calorimetry, temperature-dependent absorbance spectroscopy, and CD spectroscopy. In a buffer containing 1 m M Na cacodylate, 1 m M MgCl,, pH 6.3, the B to Z transition is centered at 76.4"C, and is characterized by AH-, = 2.02 kcal (mol base pair)-' and a cooperative unit of 150 base pairs (bp). The t, of this transition is independent of both polynucleotide and Mg2+ concentrations. A second transition, with AH,, = 2.90 cal (mol bp)-', follows the B to Z conversion, the t, of which is dependent upon both the polynucleotide and the Mg2+ concentrations. Turbidity changes are concomitant with the second transition, indicative of DNA aggregation. CD spectra recorded at a temperature above the second transition are similar to those reported for #(-)-DNA. Both the B to Z transition and the aggregation reaction are fully and rapidly reversible in calorimetric experiments. The helix to coil transition under these solution conditions is centered at 126"C, and is characterized by AHc, = 12.4 kcal (mol bp)-' and a cooperative unit of 290 bp. In 5 m M MgCl,, a single transition is seen centered at 75.5"C, characterized by AHd = 2.82 kcal (mol bp)-' and a cooperative unit of 430 bp. This transition is not readily reversible in calorimetric experiments. Changes in turbidity are coincident with the transition, and CD spectra at a temperature just above the transition are characteristic of #(-)-DNA. A transition at 124.9"C is seen under these solution conditions, with AHc, = 10.0 kcal (rnol bp)-' and which requires a complex three-step reaction mechanism to approximate the experimental excess heat capacity curve. Our results provide a direct measure of the thermodynamics of the B to Z transition, and indicate that Z-DNA is an intermediate in the formation of the #-( -) aggregate under these solution conditions.