The solvent dielectric constant is considered an important factor in determining the redox potential of the heme-containing protein cytochrome c in solution. In this study, we investigate the electrochemical response of cytochrome c in aqueous/organic solvent mixtures (100% aqueous buffer, 30% acetonitrile, 40% dimethyl sulfoxide, and 50% methanol), reporting the redox potential (Edegrees'), enthalpy, and entropy of reduction. The temperature dependence of the solvent dielectric constant (epsilon) was also measured. The results show that epsilon alone cannot regulate the Edegrees' of cytochrome c in mixed solvent systems. The implications of the temperature dependence of epsilon on the validity of the thermodynamic data are also discussed. The effect of solvent and temperature on the electron-transfer rate constant, k(s), was determined in each solvent mixture. A substantial increase in the activation energy for electron transfer was observed in 40% DMSO.