Graded doses of rat hypothalamic extract (HE) were added to dishes containing dispersed, pooled rat adenohypophyseal cells cultured for several days. ACTH secretion into the medium gave a linear log-dose response curve over a 100-fold range between 0.01 and 1 mg of NIH-HE (0.0125-1.25 rat hypothalamus). Forty percent of the maximal ACTH secretion in response to a given dose of HE occurred within 3 min. No decrease in intracellular ACTH occurred at any time or with any dose of HE, indicating that secretion was always balanced by production. ACTH secretion stopped as soon as HE-containing medium was replaced by medium without HE. The same cultured cells could be satisfactorily used in repetitive assays performed on the same or different days.