Membranes of Klebsiella pneumoniae, grown anaerobically on citrate, contain a NADH oxidase activity that is activated specifically by Na + or Li + ions and effectively inhibited by 2-heptyl-4-hydroxyquinoline-Noxide (HQNO). Cytochromes b and d were present in the membranes, and the steady state reduction level of cytochrome b increased on NaC1 addition. Inverted bacterial membrane vesicles accumulated Na + ions upon NADH oxidation. Na + uptake was completely inhibited by monensin and by HQNO and slightly stimulated by carbonylcyanidep-trifluoromethoxy phenylhydrazone (FCCP), thus indicating the operation of a primary Na + pump. A Triton extract of the bacterial membranes did not catalyze NADH oxidation by 02, but by ferricyanide or menadione in a Na +independent manner. The Na +-dependent NADH oxidation by 02 was restored by adding ubiquinone-1 in micromolar concentrations. After inhibition of the terminal oxidase with KCN, ubiquinol was formed from ubiquinone-1 and NADH. The reaction was stimulated about 6-fold by 10 mM NaC1 and was severely inhibited by low amounts of HQNO. Superoxide radicals were formed during electron transfer from NADH to ubiquinone-1. These radicals disappeared by adding NaC1, but not with NaC1 and HQNO. It is suggested that the superoxide radicals arise from semiquinone radicals which are formed by one electron reduction of quinone in a Na+-independent reaction sequence and then dismutate in a Na + and HQNO sensitive reaction to quinone and quinol. The mechanism of the respiratory Na + pump of K. pneumoniae appears to be quite similar to that of Vibrio alginolyticus.