Glycerol dehydrogenase (EC 1.1.1.6) was purified from a methylotrophic yeast, Hansenula polymorpha DL-1, for the characterisation and identification of its function. The yeast was grown in a glycerol medium. SDS-PAGE and gel permeation chromatography showed the enzyme to be composed of two subunits, each with a molecular mass of 36,000. The optimum pH for glycerol oxidation was 10. The optimum temperature for enzyme activity was 30 °C. CuCl 2 , HgCl 2 , and FeCl 3 were severely inhibitory. The activity decreased to 0% by 1 mM 2, 2'-dipyridyl and o-phenanthroline, which are inhibitors for the Fe 2+ -enzyme. An SH reagent, p-chloromercuribenzoate, was strongly inhibitory at 0.1 mM. The enzyme showed greater activity towards (R)-1,2-propanediol and (2R,3R)-2,3-butanediol than towards glycerol. No activity was detected towards 1.3-propanediol, ethanol, 1-propanol, 2-propanol, propionic acid, 1,4-butanediol, 1,2,3,4-butanetetraol, sorbitol, and L-iditol. The ratios of the activity for the R-form to that of the S-form were 5.0(R): 1(S) and 38 (R): 1(S) for 1,2-propanediol and 2,3-butanediol, respectively. The K m values for glycerol and dihydroxyacetone were 118 mM and 4.87 μM, respectively. The gene encoding the enzyme was cloned from an H. polymorpha DL-1 genomic library. Reverse transcription PCR showed that the mRNA of the enzyme was synthesised in cells grown on methanol as well as on glycerol. In the deduced amino acid sequence of 380 residues of the glycerol dehydrogenase of H. polymorpha DL-1, the NAD(H) binding pattern and the cysteine residues that correspond to the cysteine ligands at the zinc atom were conserved, as they are in sorbitol dehydrogenase, L-iditol 2-dehydrogenase, and 2,3-butanediol dehydrogenase from other origins.