This study on human cytochrome P450 reductase (CPR) presents a com-prehensive analysis of the thermodynamic and kinetic effects of pH and solvent on two- and four-electron reduction in this diflavin enzyme. pH-dependent redox potentiometry revealed that the thermodynamic equilibrium between various two-electron reduced enzyme species (FMNH • ,FADH • ; FMN,FADH2; FMNH2 ,FAD) is independent of pH.