Dolichol kinase (DK) catalyzes the CTP-dependent phosphorylation of dolichol in the biosynthesis de novo and possibly the recycling of dolichyl monophosphate in yeast and mammals. A cDNA clone from human brain encoding the mammalian homologue, hDKp, of the yeast enzyme has recently been identified. In this study hDK has been overexpressed in Chinese hamster ovary cells and shown to be a polytopic membrane protein localized in the endoplasmic reticulum with an N terminus extended into the lumen and a cytoplasmically oriented C terminus. A conserved sequence, DXXAXXXGXXXGX(8)KKTXEG, found in several enzymes utilizing CTP as substrate including DKs, phytol kinases, and several CDP-diacylglycerol synthetases has been identified, and the possibility that it is part of the CTP-binding domain of hDKp has been investigated. Topological studies indicate that the loop between transmembrane domains (TMD) 11 and TMD12 of hDKp, containing the putative CTP binding domain, faces the cytoplasm. Deletion of the loop between TMD11-12, hDK(Delta459-474), or mutation of selected conserved residues within the cytoplasmic loop results in either a partial or total loss of activity and significant reductions in the affinity for CTP. In addition, the SEC59 gene in the yeast DK mutant was sequenced, and a G420D substitution was found. Conversion of the corresponding residue Gly-443 in hDKp to aspartic acid resulted in inactivation of the mammalian enzyme. These results extend the information on the topological arrangement of hDKp and indicate that the cytoplasmic loop between TMDs 11-12, containing the critical conserved residues, lysine 470 and lysine 471 in the (470)KKTXEG(475) motif, is part of the CTP-binding site in hDK.