We have determined the formal potential values for each electron transfer to electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO), in order to further characterize the thermodynamics of electron transport from various acyl-CoA thioesters to the mitochondrial ubiquinone pool. ETF-QO contains one [4Fe-4S]2+,1+ cluster and one FAD prosthetic group. A preliminary visible-spectroelectro-chemical titration showed that the two redox centers were reduced almost simultaneously. Since the visible spectra of the chromophores overlap, it was not possible to resolve the formal potential value for each electron transfer to the protein using this method. Accordingly, an EPR-spectroelectrochemical cell was designed so that each formal potential value could be resolved by EPR quantitation of the flavin semiquinone and the reduced iron-sulfur cluster during the titration. The formal potential values for electron transfer to ETF-ubiquinone oxidoreductase at pH 7.5 and 4 °C were E1°ʹ = +0.028 V and E2°ʹ = -0.006 V for the first and second electron transfers, respectively, to the FAD and E°ʹ = +0.047 V for the iron-sulfur cluster. The thermodynamics of electron transport from the acyl-CoA substrates of β-oxidation to the mitochondrial electron transport chain have been fully resolved with completion of this work. The results are discussed in terms of their significance to the overall electron transport process from β-oxidation.