The Na+ -translocating NADH:quinone oxidoreductase Na+-NQR) from the human pathogen Vibrio cholerae is a respiratory flavo-FeS complex composed of the six subunits NqrA-F. The Na+-NQR was produced as His6-tagged protein by homologous expression in V. cholerae. The isolated complex contained near stoichiometric amounts of non-covalently bound FAD (0.78 mol/mol Na+-NQR) and riboflavin (0.70 mol/mol Na+-NQR), catalyzed NADH-driven Na+ transport (40 nmol Na+ min-1 mg-1), and was inhibited by 2-n-heptyl-4-hydroxyquinoline-N-oxide. EPR spectroscopy showed that Na+-NQR as isolated contained very low amounts of a neutral flavosemiquinone (10-3 mol/mol Na+-NQR). Reduction with NADH resulted in the formation of an anionic flavosemiquinone (0.10 mol/mol Na+-NQR). Subsequent oxidation of the Na+-NQR with ubiquinone-1 or O2 led to the formation of a neutral flavosemiquinone (0.24 mol/mol Na+-NQR). We propose that the Na+-NQR is fully oxidized in its resting state, and discuss putative schemes of ADH-triggered redox transitions.