ACTH release from the anterior pituitary gland is principally driven by the two hypothalamic hormones, corticotropin- releasing hormone (CRH) and arginine vasopressin (AVP). Using the reverse hemolytic plaque assay, we have compared the effects of CRH and AVP on ACTH release from individual, dispersed pituitary cells. A small percent (0.36 ± 0.06%) of pituitary cells formed plaques when exposed to medium alone. AVP caused 3.44 ± 0.10% of cells to form plaques (P < 0.01 compared with medium alone), CRH produced 4.85 ± 0.20% plaque-forming cells (P < 0.01 compared with AVP), and the combination of CRH and AVP produced a still greater percent of plaque-forming cells (5.80 ± 0.20%, P < 0.01 compared with CRH alone). A double reverse hemolytic plaque assay was then employed to examine whether some cells formed plaques only in the presence of one or other secretagogue. Using this technique we found clear evidence of cells that formed plaques in response to CRH but not AVP (P < 0.005); CRH or AVP (P < 0.0001), and CRH and AVP (P < 0.05). There was no evidence of a corticotrope forming a plaque with AVP but not CRH (P = 0.52). Thus there appears to be functionally distinct classes of corticotropes. These findings have important implications for our understanding of the relative responsiveness of the pituitary to hypothalamic secretagogues and provide a new physiological perspective on recent reports of stress-specific hypothalamic responses regulating ACTH release. (Endocrinology128: 197–203, 1991)