A pancreatic islet-specific glucose-6-phosphatase-related protein (IGRP) was cloned using a subtractive cDNA expression cloning procedure from mouse insulinoma tissue. Two alternatively spliced variants that differed by the presence or absence of a 118-bp exon (exon IV) were detected in normal balb/c mice, diabetic ob/ob mice, and insulinoma tissue. The longer, 1901-bp full-length cDNA encoded a 355-amino acid protein (molecular weight 40,684) structurally related (50% overall identity) to the liver glucose-6-phosphatase and exhibited similar predicted transmembrane topology, conservation of catalytically important residues, and the presence of an endoplasmic reticulum retention signal. The shorter transcript encoded two possible open reading frames (ORFs), neither of which possessed His174, a residue thought to be the phosphoryl acceptor (Pan CJ, Lei KJ, Annabi B, Hemrika W, Chou JY: Transmembrane topology of glucose-6-phosphatase. J Biol Chem 273:6144-6148, 1998). Northern blot and reverse transcription-polymerase chain reaction analysis showed that the mRNA was highly expressed in pancreatic islets and expressed more in beta-cell lines than in an alpha-cell line. It was notably absent in tissues and cell lines of non-islet neuroendocrine origin, and no other major tissue source of the mRNA was found. During development, it was expressed in parallel with insulin mRNA. The mRNA was efficiently translated and glycosylated in an in vitro translation/membrane translocation system and readily transcribed into COS 1, HIT, and CHO cells using cytomegalovirus or Rous sarcoma virus promoters. Whereas the liver glucose-6-phosphatase showed activity in these transfection systems, the IGRP failed to show glucose phosphotransferase or phosphatase activity with p-nitrophenol phosphate, inorganic pyrophosphate, or a range of sugar phosphates hydrolyzed by the liver enzyme. While the metabolic function of the enzyme is not resolved, its remarkable tissue-specific expression warrants further investigation, as does its transcriptional regulation in conditions where glucose responsiveness of the pancreatic islet is altered.