Lipoamide dehydrogenase has been isolated from acid-precipitated human liver particles in a highly purified state by a freezing and thawing technique instead of the heat treatment used by other workers. The enzyme was found to have a molecular weight of 138,000,and to contain 2 moles of FAD per mole of protein. In these respects, as in substrate specificity, Michaelis constant, absorption spectrum, and kinetic parameters, the enzyme is similar to preparations isolated from beef liver, pig heart and bacteria. This enzyme catalyzed the reduction of NAD by dihydrolipoamide and exhibited diaphorase and transhydrogenase activities. If the enzyme was incubated with the dithiol inhibitors such as arsenite and cadmium chloride in the presence of NADH_2,lipoamide dehydrogenase and transhydrogenase activities were strongly inhibited, but diaphorase activity was enhanced.