Only one type (membrane-bound form) of NADH dehydrogenase could be detected in the log-phase cells ofBacillus megaterium. By sonification this enzyme could be effectively solubilized, while NADH oxidase remained bound to the membrane. A molecular weight of about 40 Kd was estimated for the dehydrogenase by gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) with an activity stain. Mercuric chloride and 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO) were inhibitors for both the NADH dehydrogenase and oxidase inB. megaterium. The inhibition studies of NADH oxidation suggested that NADH dehydrogenase provided the primary electron source for NADH oxidase in this organismin vitro. NADH dehydrogenase was highly specific for NADH, and Km was estimated to be 28.2 μM. The enzyme was subjected to end-product inhibition of a competitive type.