The inositol-polyphosphate 5-phosphatase enzyme family removes the 5-position phosphate from both inositol phosphate and phosphoinositide signaling molecules. We have cloned and characterized a novel 5-phosphatase, which demonstrates a restricted substrate specificity and tissue expression. The 3.9-kb cDNA predicts for a 72-kDa protein with an N-terminal proline rich domain, a central 5-phosphatase domain, and a Cterminal CAAX motif. The 3.9-kilobase mRNA showed a restricted expression but was abundant in testis and brain. Antibodies against the sequence detected a 72-kDa protein in the testis in the detergent-insoluble fraction. Indirect immunofluorescence of the Tera-1 cell line using anti-peptide antibodies to the 72-kDa 5-phosphatase demonstrated that the enzyme is predominantly located to the Golgi. Expression of green fluorescent protein-tagged 72-kDa 5-phosphatase in COS-7 cells revealed that the enzyme localized predominantly to the Golgi, mediated by the N-terminal proline-rich domain, but not the C-terminal CAAX motif. In vitro, the protein inserted into microsomal membranes on the cytoplasmic face of the membrane. Immunoprecipitated recombinant 72-kDa 5-phosphatase hydrolyzed phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,5-bisphosphate, forming phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3-phosphate, respectively. We propose that the novel 5-phosphatase hydrolyzes phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,5-bisphosphate on the cytoplasmic Golgi membrane and thereby may regulate Golgi-vesicular trafficking.The inositol-polyphosphate 5-phosphatases (5-phosphatases) 1 are a large family of enzymes that remove the 5-position phosphate from the inositol ring of phosphatidylinositols including phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ), phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ), and the inositol phosphates, inositol 1,4,5-trisphosphate (Ins(1,4,5)-P 3 ) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P 4 ). Nine distinct mammalian 5-phosphatases have been identified and characterized, while four 5-phosphatases have been described in Saccharomyces cerevisiae. All 5-phosphatases are defined by the presence of a conserved 300-amino acid domain, which contains two signature motifs, proposed to mediate substrate binding and catalysis (1, 2). Although all 5-phosphatase enzymes contain these signature motifs, there is considerable diversity in the substrate specificity of 5-phosphatase isoforms. The enzyme family has been subclassified on this basis into four types, I-IV. The type I enzymes, characterized by the 43-kDa 5-phosphatase (also called 5-phosphatase I), hydrolyze Ins(1,4,5)P 3 and Ins(1,3,4,5)P 4 but not any of the 5-position phosphoinositide substrates; the type II 5-phosphatases, including synaptojanin, 5-phosphatase II (originally designated the 75-kDa 5-phosphatase), and the protein product of the oculocerebrorenal syndrome (OCRL) gene, hydrolyze PtdIns-(4,5)P 2 , PtdIns(3,4,5)P 3 , Ins(1,3,4,5)P 4 ...