Two kinds of NADH oxidases were isolated from the respiratory chain of Vibrio alginolyticus in order to study their enzymatic properties. Wild type membranes, which possess NADH : quinone reductase 2 (NQR2), as well as NADH : quinone reductase 1 (NQR1), oxidized both NADH and deamino-NADH as substrntes. Membranes of a mutant bacterium defective in the Na+ pump (Nap1), which contain only NQR2, oxidized NADH exclusively. Compared with NQR1 the reductase activity of NQR2 for artificial electron acceptors in the presence of NADH or deamino-NADH was relatively low. These two reductases have different specificities for electron acceptors. Wild type membranes exhibited two apparent K_m values for NADH, 5 to 8 μM and 25 to 30 μM. Napl membranes exhibited only a single apparent K_m value of 27 to 35 μM for NADH. The two kinds of NADH oxidase in the V. alginolyticus respiratory chain apparently differ considerably in their enzymatic properties.