A soluble enzyme that catalyzes the transfer of D-glucose from UDP-D-glucose to dolichyl phosphate has been prepared by sonic oscillation of Acanthamoeba castellani cysts. The product of catalysis is dolichyl beta-D-glucosyl phosphate. The enzyme requires a divalent cation, either magnesium or manganese, and the presence of a reducing agent for maximum activity. Solanesyl phosphate and ficaprenyl phosphate are alternative substrates, apparently at lower rates, but GDP-D-glucose, UDP-D-glucuronic acid, UDP-N-acetyl-D-glucosamine, and UDP-D-xylose are not substrates. The temperature optimum is 30 degrees C, the pH optimum is pH 7.0, the Km for UDP-Glc is 9.1 microM and for dolichyl phosphate it is 4.5 microM. Uridine monophosphate and UDP are inhibitors of the reaction, UDP causing reversal and UMP being a competitive inhibitor of UDP-Glc with a Ki of 62 microM. The enzyme can be stored indefinitely below -20 degrees C, is stable for several days at 4 degrees C, but is half-inactivated within 2 h at 30 degrees C and completely inactivated within 10 min at 52 degrees C.