The properties of NADP+-linked glycerol dehydrogenase (EC 1 . 1 . 1 .72) from Neurospora crassa were studied following 505-fold purification, with 54% yield, by gel filtration, ion exchange chromatography and affinity chromatography. Specific staining for the purified enzyme after disc gel electrophoresis revealed a single band and after isoelectric focusing, five bands. No enzymically inactive bands were detected by general protein staining in control gels. Total molecular weight was estimated to be about 160000, with a subunit molecular weight of about 43 000. The forward reaction from glycerol to D-glyceraldehyde had a pH optimum of 9.5 and was specific for glycerol as substrate, since no activity was detected with other polyols tested. The reverse reaction had a pH optimum of 6.5 and was maximal with D-glyceraldehyde as substrate. K , values for glycerol and D-glyceraldehyde were 1.43 x 10-1 M and 1.15 x M, respectively. Dihydroxyacetone also served as substrate in the reverse reaction. The enzyme was NADP+-specific.