Mannosylphosphodolichol synthase (MPD-synthase) (EC 2.4.1.830) catalyzing formation of MPD from GDPMan and dolichylphosphate (PD) has been purified from T. reesei cellular membranes almost to homogeneity. Selective solubilization of the enzyme was followed by one step purification on Phenyl-Sepharose column. SDS/ PAGE of the purified enzyme fraction revealed the presence of a protein band of 31 kDa corresponding to the apparent molecular mass of the MPD-synthase purified from S. cerevisiae [Babczinski, P. et al. (1980) Eur. J. Biochem. 105, 509-515; Haselbeck A. (1989) Eur. J. Biochem. 181, 663-668]. During solubilization, the enzyme was stabilized by the presence of a lipophilic substrate dolichylphosphate and phospholipids as well as by protease inhibitors. The Phenyl-Sepharose purified enzyme had an absolute requirement for dolichylphosphate and was activated by cAMP dependent protein kinase.