D-Aspartate (D-Asp) is found in prolactin (PRL)containing cells of the rat anterior pituitary gland [Lee et al., Brain Res. 838, 193-199, 1999]. In order to determine whether D-Asp is actually produced by the anterior pituitary gland and whether it plays a physiological role in PRL function, a PRL-secreting clonal strain of rat pituitary tumor cells (GH(3)) was employed in this study. HPLC analysis and immunocytochemical staining detected the presence and synthesis of D-Asp in the cytoplasm of these cells. In addition, thyrotropin-releasing hormone-stimulated PRL secretion was increased in a dose-dependent fashion by D-Asp from these cells. These results suggest that the anterior pituitary gland synthesizes D-Asp and that D-Asp acts as a messenger in this gland,