We searched for a source of reactive oxygen species (ROS) in the cytoplasm of the human pathogen Vibrio cholerae and addressed the mechanism of ROS formation using the dye 2=,7=-dichlorofluorescein diacetate (DCFH-DA) in respiring cells. By comparing V. cholerae strains with or without active Na ؉ -translocating NADH:quinone oxidoreductase (Na ؉ -NQR), this respiratory sodium ion redox pump was identified as a producer of ROS in vivo. The amount of cytoplasmic ROS detected in V. cholerae cells producing variants of Na ؉ -NQR correlated well with rates of superoxide formation by the corresponding membrane fractions. Membranes from wild-type V. cholerae showed increased superoxide production activity (9.8 ؎ 0.6 mol superoxide min ؊1 mg ؊1 membrane protein) compared to membranes from the mutant lacking Na ؉ -NQR (0.18 ؎ 0.01 mol min ؊1 mg ؊1 ). Overexpression of plasmid-encoded Na ؉ -NQR in the nqr deletion strain resulted in a drastic increase in the formation of superoxide (42.6 ؎ 2.8 mol min ؊1 mg ؊1 ). By analyzing a variant of Na ؉ -NQR devoid of quinone reduction activity, we identified the reduced flavin adenine dinucleotide (FAD) cofactor of cytoplasmic NqrF subunit as the site for intracellular superoxide formation in V. cholerae. The impact of superoxide formation by the Na ؉ -NQR on the virulence of V. cholerae is discussed. IMPORTANCEIn several studies, it was demonstrated that the Na ؉ -NQR in V. cholerae affects virulence in a yet unknown manner. We identified the reduced FAD cofactor in the NADH-oxidizing NqrF subunit of the Na ؉ -NQR as the site of superoxide formation in the cytoplasm of V. cholerae. Our study provides the framework to understand how reactive oxygen species formed during respiration could participate in the regulated expression of virulence factors during the transition from aerobic to microaerophilic (intestinal) habitats. This hypothesis may turn out to be right for many other pathogens which, like V. cholerae, depend on the Na ؉ -NQR as the sole electrogenic NADH dehydrogenase. Vibrio cholerae is a Gram-negative bacterium and the causative agent of cholera, a devastating diarrheal disease. It is found in estuaries, inhabiting seawater or brackish water where it is commonly associated with biofilms on phyto-or zooplankton (1). The high salinity in these habitats is counteracted by an electrochemical sodium motive force (SMF) generated by the respiratory chain of V. cholerae (2, 3). This SMF drives cellular processes like substrate transport (4) and motility (5). It is generated by the Na ϩ -translocating NADH:quinone oxidoreductase (Na ϩ -NQR), which couples the energy that is released by oxidation of NADH with ubiquinone to the transport of sodium ions out of the cytoplasm with a 1e Ϫ /1Na ϩ stoichiometry (6). The Na ϩ -NQR is a membrane-bound protein complex consisting of six subunits, NqrABCDEF, and contains a variety of different cofactors (3). Electron transfer from NADH to ubiquinone is mediated by a noncovalently bound flavin adenine dinucleotide (FAD) and a [2Fe-2...