Tetrahymena pyriformis contains an enzyme which hydrolyzed dolichyl phosphate. This activity was solubilized from lyophilized samples of this organism and was relatively stable when stored frozen. The soluble enzyme preparation had an acid pH optimum and hydrolyzed both dolichyl and phytanyl phosphates at equivalent rates. The polyprenylphosphate phosphatase activity was compared with the acid phosphatases which hydrolyzed p-nitrophenyl phosphate and marked differences were found. Dolichyl phosphate hydrolysis required Mg2+ for maximum activity while the bulk of the phosphatase activity was not effected by the absence of this ion. Other differences were that the polyprenylphosphate phosphatase was relatively insensitive to inhibitors such as tartrate and vanadium oxide sulfate which had a pronounced effect on the rate of p-nitrophenyl phosphate hydrolysis. The two activities also appeared to have different subcellular distributions. The polyprenylphosphate phosphatase was markedly inhibited by ethoxy formic anhydride, a reagent which is active against enzymes containing a histidine residue at their active site, while p-nitrophenyl phosphate hydrolysis was unaffected. The polyprenylphosphate phosphatase may be important in regulating the level of dolichyl phosphate in T. pyriformis and thus the rate of glycoprotein synthesis. It is also a useful tool which is capable of liberating dolichol from dolichyl phosphate under mild conditions which will permit the further characterization of the polyprenols.