Abstract The glycerol kinase (EC 27.1.30) of the salt-tolerant yeast Debaryomyces hansenii was purified approx. 3300-fold by a combination of chromatographic procedures. The specific activity of the final preparation was 177 units per mg protein and the yield about 21%. The elution profile from the final Mone Q column indicated that the enzyme preparation was not completely homogeneous, as was further confirmed by silver-stained SDS-polyacrylamide gels. A molecular weight of 236000 for the catalytically active enzyme was estimated by gel filtration. KCl stimulated the enzyme activity slightly; other salts acted as inhibitors. The pH optimum was at 7.0–8.0. The enzyme phosphorylated glycerol exclusively and ATP was the most effective phosphoryl group donor tested. The K m ATP was estimated to be 0.5 mM and K m glycerol to be 5 mM. ADP acted as a competitive inhibitor with respect to ATP; the K i was estimated as 0.5 mM. Fructose 1,6-biphosphate was only slightly inhibitory to the purified enzyme. The kinetic properties of the enzyme suggest that the glycerol kinase functions in catalyzing the exit reaction from the osmoregulatory glycerol pool.