An NAD-dependent alcohol-aldehyde oxidoreductase was purified to homogeneity and characterized from cell extracts of the thermophilic microorganism Bacillus acidocaldarius. The 500-fold purified homogeneous enzyme had a molecular mass of 154 kDa, as shown by gel filtration and glycerol gradient centrifugation. On sodium dodecyl sulfate polyacrylamide gel electrophoresis the protein showed one band of 38 kDa, indicating that the enzyme is a tetramer composed of subunits of identical molecular weight. Ethanol was the best substrate with the highest kcat/Km values, and the enzyme showed a substrate specificity that included linear, secondary and cyclic alcohols, as well as anisaldehyde, but it was not active on ketones. The protein contains eight zinc atoms per tetramer, four of which are removed by chelating agents with a concomitant loss of thermal stability. Circular dichroism spectra and determination of the NH2-terminal sequence allowed structural and homology comparison with other alcohol dehydrogenases from animal and bacterial sources.