We have measured the temperature dependence of the oxygen-binding isotherms of human and bovine hemoglobin at pH 9.0 in 0.1 M borate buffer. In both hemoglobins the ionization of the Bohr protons is finished at this pH; therefore, their heat does not interfere with the measurements. Two sets of curves have been obtained, which have been analyzed by either singular or global procedures for estimating the enthalpy changes of subsequent steps of oxygenation. The data indicate that in human hemoglobin the reaction with oxygen is enthalpy driven for steps 1, 2, and 4 while it is entropy driven for step 3. In bovine hemoglobin this phenomenon is even more evident: steps 2 and 4 are enthalpy driven while steps 1 and 3 are entropy driven. The discontinuous distribution of heat at subsequent steps of oxygenation suggests that the T to R transition in hemoglobin is not a monotonic process and involves conformations with novel characteristics.