Euglena gracilis is a freshwater free-living organism able to grow with ethanol as carbon source; to facilitate this metabolism several alcohol dehydrogenase (ADH) activities have been detected. We report the gene cloning, over-expression, and biochemical characterization of a medium-chain NAD(+)-dependent ADH from E. gracilis (EgADH). The enzyme's amino acid sequence displayed the highest percentages of similarity and identity with ADHs of bacteria and fungi. In the predicted three-dimensional model, all the residues involved in Zn(2+), cofactor, and substrate binding were conserved. A conventional signal peptide for import into mitochondria could not be clearly identified. The protein of 37 kDa was over-expressed, purified to homogeneity, and kinetically characterized. The enzyme's optimal pH was 7.0 for ethanol oxidation displaying a V(m) of 11.7+/-3.6 U/mg protein and a K(m) of 3.2+/-0.7 mM for this substrate. Isopropanol and isopentanol were also utilized, although with less efficiency. It showed specificity for NAD(+) with a K(m) value of 0.39+/-0.1 mM and Mg(2+) or Zn(2+) were essential for activity. The recombinant EgADH reported here may help to elucidate the roles that different ADHs have on the metabolism of short- and long-chain alcohols in this microorganism.