Pig liver electron-transfer flavoprotein-ubiquinone oxidoreductase has been investigated by room temperature UV-visible, low-temperature electron paramagnetic resonance and low-temperature magnetic circular dichroism spectroscopies. The results provide unambiguous evidence for the presence of a single [4Fe-4S] cluster that is diamagnetic in the isolated enzyme and becomes paramagnetic with an S = 1/2 ground state on reduction with dithionite or enzymatically with the physiological electron donor. The EPR data for samples at pH 7.8 indicate that FAD is reduced by one electron to the anionic semiquinone form in the enzymatically reduced enzyme, and by two electrons to the hydroquinone form by excess dithionite. The possibility of weak spin-spin interaction between the FAD semiquinone and the [4Fe-4S]1+ center is discussed in the light of the observation of a small increase in the linewidth of the Fe-S EPR in enzymatically reduced samples.