High NAD-dependent alcohol dehydrogenase (ADH) activity was found in the cytoplasm when a membrane-bound, quinoprotein, ADH-deficient mutant strain of Acetobacter pasteurianus SKU1108 was grown on ethanol. Two NAD-dependent ADHs were separated and purified from the supernatant fraction of the cells. One (ADH I) is a trimer, consisting of an identical subunit of 42 kDa, while the other (ADH II) is a homodimer, having a subunit of 31 kDa. One of the two ADHs, ADH II, easily lost the activity during the column chromatographies, which could be stabilized by the addition of DTT and MgCl2 in the column buffer. ADH I but not ADH II contained approximately one zinc atom per subunit. The N-terminal amino acid analysis indicated that ADH I and ADH II have homology to the long-chain and short-chain ADH families, respectively. ADH I showed a preference for primary alcohols, while ADH II had a preference for secondary alcohols. The two ADHs showed clear difference in their kinetics on ethanol, acetaldehyde, NAD, and NADH. The physiological function of both ADH I and ADH II are also discussed.