The Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) is present in the membranes of a number of marine bacteria and pathogenic bacteria. Two of the six subunits of the Na(+)-NQR, NqrB and NqrC, have been previously shown to contain covalently bound flavin adenine mononucleotide (FMN). In the current work, the cloning of nqrC from Vibrio cholerae is reported. The gene has been expressed in V. cholerae and shown to contain one equivalent of covalently bound FMN. In contrast, no covalent flavin was detected when threonine-225 was replaced by leucine. The data show that the FMN attachment does not require assembly of the enzyme and are consistent with the unusual threonine attachment site.