Intramolecular electron transfer in azurin in water and deuterium oxide has been studied over a broad temperature range. The kinetic deuterium isotope effect, k _H / k _D , is smaller than unity (0.7 at 298 K), primarily caused by the different activation entropies in water (−56.5 J K ⁻¹ mol ⁻¹ ) and in deuterium oxide (−35.7 J K ⁻¹ mol ⁻¹ ). This difference suggests a role for distinct protein solvation in the two media, which is supported by the results of voltammetric measurements: the reduction potential ( E ^0′ ) of Cu ^2+/+ at 298 K is 10 mV more positive in D ₂ O than in H ₂ O. The temperature dependence of E ^0′ is also different, yielding entropy changes of −57 J K ⁻¹ mol ⁻¹ in water and −84 J K ⁻¹ mol ⁻¹ in deuterium oxide. The driving force difference of 10 mV is in keeping with the kinetic isotope effect, but the contribution to Δ S ^‡ from the temperature dependence of E ^0′ is positive rather than negative. Isotope effects are, however, also inherent in the nuclear reorganization Gibbs free energy and in the tunneling factor for the electron transfer process. A slightly larger thermal protein expansion in H ₂ O than in D ₂ O (0.001 nm K ⁻¹ ) is sufficient both to account for the activation entropy difference and to compensate for the different temperature dependencies of E ^0′ . Thus, differences in driving force and thermal expansion appear as the most straightforward rationale for the observed isotope effect.