Isolated rat anterior pituitary cells have been prepared by mechanical agitation of quartered glands in the presence of trypsin. The cells appear morphologically and functionally intact and contain large amounts of ACTH. The medium separated from cells incubated at 37° for 20 min contains small but significant amounts of ACTH. Acetic acid extracts of rat HME or cerebral cortical tissue and arginine vasopressin increase the ACTH content of the medium.