The complete amino acid sequence of alcohol dehydrogenase of Thermoanaerobium brockii (TBAD) is presented. The S-carboxymethylated protein was cleaved at methionine residues (with cyanogen bromide) to provide a set of 10 nonoverlapping fragments accounting for 90% of the sequence. These fragments were then overlapped and aligned, and the sequence was completed by using peptides generated by proteolytic cleavage at lysine residues (with Achromobacter protease I). The protein subunit contained 352 amino acid residues corresponding to a molecular weight of 37,652. The sequence showed about 35% identity with that of the prokaryotic Alcaligenes eutrophus alcohol dehydrogenase and about 25% identity with any one of the eukaryotic alcohol/polyol dehydrogenases known today. Of these, only 18 residues (5%) are strictly conserved: 11 Gly, 2 Asp, and 1 each of Cys, His, Glu, Pro, and Val.