The energy-converting NADH:ubiquinone oxidoreductase, also known as respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of ions across the membrane. It was assumed that the complex exclusively works as a proton pump. Recently, it has been proposed that complex I from Klebsiella pneumoniae and Escherichia coli work as Na+ pumps. We have used an E. coli complex I preparation to determine the type of ion(s) translocated by means of enzyme activity, generation of a membrane potential and redox-induced Fourier-transform infrared spectroscopy. We did not find any indications for Na+ translocation by the E. coli complex I.