Circular dichroism has been used to investigate the thermodynamics of the thermal unfolding of Escherichia coli ribonuclease HI as a function of pH over the pH range 0-4. This protein undergoes a reversible thermal conformational change from the native state to the denatured state with an isodichroic point. The calculated thermodynamic values at pH 3.0 are as follows : t(m) 50.2 degrees C, Delta H-m (t(m)) = 93.8 kcal mol(-1), Delta G (25 degrees C) = 6.08 kcal mol(-1), and Delta G (10 degrees C) = 8.14 kcal mol(-1). At pH 4, Delta G (25 degrees C) = 10 kcal mol(-1) and Delta G (10 degrees C) = 12 kcal mol(-1). At a pH below 2, this protein denatures at 25 degrees C with Delta G (25 degrees C) = -1 kcal mol(-1), but it is stable at 10 degrees C with Delta G (10 degrees C) = 2 kcal mol(-1). The Delta C-p value determined from the Delta H-m(T-m) versus T-m plot is 1.4 kcal mol(-1) K-1. The thermal unfolding curves at pH values above 2.18 showed a highly cooperative thermal transition. Cold denaturation was observed at temperatures below 10 degrees C between pH 2.03 and 1.55. A cooperative heat denaturation was also observed over this pH range. At pH values lower than 1.45, cold denaturation was not observed. A broad thermal transition was observed between pH 0.83 and 0.53.