The cellular localization of D: -alanine (D: -Ala) in the rat pituitary gland, the tissue containing the highest amount of D: -Ala, has been clarified for the first time by enantioselective visualization of D: -Ala using our own established mouse monoclonal antibody against D: -Ala. D: -Ala immunopositive cells were present predominantly in the anterior lobe, while no intense staining was observed in the intermediate and posterior lobes. The anterior pituitary gland contains five types of cells secreting specific hormones (growth hormone, adrenocorticotropic hormone (ACTH), gonadotropic hormone, prolactin, and thyroid-stimulating hormone), and the double staining results indicated that D: -Ala is localized to the ACTH-secreting cells. The localization of D: -Ala is clearly different from that of D: -aspartic acid (D: -Asp), which is observed in the prolactin cells. Considered together with our previous findings that D: -Ala is localized to the insulin-secreting beta-cells in the pancreas, and both ACTH and insulin are typical regulatory hormones of blood glucose, D: -Ala is suggested to have some functional relationships to blood glucose level regulation in mammals.