Regenerating gene (Reg), first isolated from a regenerating islet cDNA library, encodes a secretory protein with a growth stimulating effect on pancreatic beta cells that ameliorates the diabetes of 90% depancreatized rats and non-obese diabetic mice. Reg and Reg-related genes have been revealed to constitute a multigene family, the Reg family, which consists of three subtypes (types I, II, III) based on the primary structures of the encoded proteins of the genes. We have isolated three types of mouse Reg family gene (Reg I, Reg II, Reg IIIalpha, Reg IIIbeta and Reg IIIgamma) [Unno et al. (1993) J. Biol. Chem. 268, 15974-15982; Narushima et al. (1997) Gene 185, 159-168]. In the present study, by Southern blot analysis of a mouse bacterial artificial chromosome clone containing the five Reg family genes in combination with PCR cloning of every interspace fragment between adjacent genes, the Reg family genes were mapped to a contiguous 75kb region of the mouse genome according to the following order: 5'-Reg IIIbeta-Reg IIIalpha-Reg II-Reg I-Reg IIIgamma-3'. In the process of ordering the genes, we sequenced the 6.8kb interspace fragment between Reg IIIbeta and Reg IIIalpha and encountered a novel type III Reg gene, Reg IIIdelta. This gene is divided into six exons spanning about 3kb, and encodes a 175 amino acid protein with 40-52% identity with the other five mouse Reg (regenerating gene product) proteins. Reg IIIdelta was expressed predominantly in exocrine pancreas, but not in normal islets, hyperplastic islets, intestine or colon, whereas both Reg I and Reg II were expressed in hyperplastic islets and Reg IIIalpha, Reg IIIbeta and Reg IIIgamma were expressed strongly in the intestinal tract. Possible roles of Reg IIIdelta and the widespread occurrence of the Reg IIIdelta gene in mammalian genomes are discussed.