A full-length E(ecto)-ATPase (Plesner, L. (1995) Int. Rev. Cytol. 158, 141-214) cDNA was cloned from a human brain cDNA library; it encodes a 610-amino acid protein that contains two putative transmembrane domains. Heterologous expression of this protein in COS-7 cells caused a significant increase in intracellular membrane-bound nucleoside phosphatase activity. The activity was highest with UDP as substrate and was stimulated by divalent cations in the following order: Ca2+ >> Mg2+ > Mn2+. The results of immunofluorescence staining indicate that this protein is located in the Golgi apparatus. UDP hydrolysis was increased in the presence of Triton X-100 or alamethicin, an ionophore that facilitates movement of UDP across the membrane, suggesting that the active site of this UDPase is on the luminal side of the Golgi apparatus. This is the first identification of a mammalian Golgi luminal UDPase gene. Computer-aided sequence analysis of the EATPase superfamily indicates that the human UDPase is highly similar to two hypothetical proteins of the nematode Caenorhabditis elegans and to an unidentified 71.9-kDa yeast protein and is less related to the previously identified yeast Golgi GDPase.