The distribution of high affinity histamine H3 receptors in various tissues from guinea pig has been determined using [3H]N alpha-methylhistamine binding. In the course of those studies, it was observed that the pituitary gland contains H3 receptors. Using this radioligand, we have now identified and characterized H3 receptors on' the AtT-20 cell line from a murine anterior pituitary tumor. This line has approximately 5000 high affinity (KD = 0.7 nM) H3 binding sites per cell. Competition binding with standard H1, H2 and H3 agents has confirmed that these sites are, indeed, H3 receptors. The H3 receptor specific agonist, (R)-alpha-methylhistamine increased the release of adrenocorticotropic hormone (ACTH) from AtT-20 cells in a dose- and time-dependent manner, while histamine and the H2 agonist dimaprit were significantly less potent. Furthermore, this response was blocked by thioperamide, an H3 receptor specific antagonist, but not by the H1 and H3 antagonists, chlorpeniramine and cimetidine. These results identify, for the first time, a cell line expressing H3 receptors and indicate that the high affinity histamine H3 receptor regulates ACTH release from that cell.