The NAD(+)-dependent alcohol dehydrogenase (EC 1.1.1.1) from the thermoacidophilic archaebacterium Sulfolobus solfataricus, DSM1617 strain (SSADH), has been purified and characterized. Its gene has been isolated by screening two S. Solfataricus genomic libraries using oligonucleotide probes. The encoding sequence consists of 1041 base pairs, and it shows a high preference for codons ending in T or A. The primary structure, determined by peptide and gene analysis, consists of 347 amino acid residues, yielding a molecular weight of 37,588. A level of identity of 24-25% was found with the amino acid sequences of horse liver, yeast, and Thermoanaerobium brockii alcohol dehydrogenases. The coenzyme-binding and catalytic and structural zinc-binding residues typical of eukaryotic alcohol dehydrogenases were found in SSADH with the difference that one out of the four structural zinc-binding Cys residues is substituted by Glu. The protein contains four zinc atoms per dimer, two of which are removed by chelating agents with a concomitant loss of structural stability.