A mouse monoclonal antibody against D-alanine (D-Ala) has been raised and the immunohistochemical localization of this D-amino acids in the rat pancreas is visualized. The obtained anti-D-Ala monoclonal antibody has no significant cross-reactivity to all proteino-genic L-amino acids and their D-enantiomers. Using this antibody, immunohistochemical staining was performed on the pancreas, and specific staining for D-Ala has been observed only in the Langerhans islets. To identify the types of D-Ala-immunopositive cells, double staining was carried out with antibodies against D-Ala and pancreatic hormones. Similar immunostaining patterns have been observed for D-Ala and insulin, while D-Ala is hardly co-localized with other hormones (glucagon, somatostatin, and pancreatic polypeptide). These results indicate for the first time that D-Ala is localized to insulin producing P-cells in mammalian pancreas, suggesting that this D-amino acid would be involved in the regulation of the blood glucose level. (c) 2007 Published by Elsevier Inc.