The purpose of the present experiments was firstly, to examine the efficacy of the oxycellulose-Amberlite CG-50 extraction procedure for the isolation of rat pituitary ACTH and secondly, to study the effects of both hypothalamic extract and corticosterone on the synthesis of rat pituitary ACTH. Briefly, ‘cold’ rat pituitary ACTH was monitored by bioassay in each step of the isolation procedure as a function of corticosterone stimulation in incubated adrenal slices. Labeled ACTH was isolated from rat pituitaries previously incubated in vitro and obtained from adrenalectomized animals and cortisone acetate treated animals to provide further data to substantiate that the labeled isolated protein contained ACTH. Cold ACTH was detected in two purified fractions (oxycellulose and Amberlite CG-50). Adrenalectomy stimulated and cortisone acetate suppressed the incorporation of <sup>14</sup>C-phenylalanine into the ACTH-like protein in these two fractions. A continuously increasing uptake of the label into ACTH-like protein in the isolated fractions occurred with time providing evidence that de novo synthesis had taken place. Corticosterone inhibited ACTH synthesis at both 10<sup>-4</sup> and 10<sup>-6</sup> M, whereas, hypothalamic stalk median eminence (HSME) caused a significant, but not dose-related, increase in ACTH synthesis with <sup>3</sup>/<sub>5</sub> 1, and 2 HSME equivalents added.