A method has been developed for studying the rate of in vivo ACTH synthesis by measuring the incorporation of phenylalanine-¹⁴C into ACTH in the rat. At 1 h after the intra-peritoneal injection of labeled amino acid the anterior pituitary was obtained and ACTH was extracted with hot glacial acetic acid, precipitated with ether, and adsorbed onto oxidized cellulose. The ACTH fraction was then eluted with 0.1 N hydrochloric acid. This fraction was shown to contain radioactive ACTH and did not contain free phenylalanine-¹⁴C. Changes in the rate of ACTH synthesis were estimated by changes in specific activity of the ACTH following a 1 h exposure of the rat to isotope. At 7, 14, and 24 days following adrenalectomy the rate of pituitary ACTH labeling doubled, while at 40 days, there was a three- to fivefold increase over the control rate. Administration of cortisol to the adrenalectomized but not to the intact rat decreased the rate of pituitary ACTH labeling. Nearly total inhibition of ACTH synthesis was caused by cortisol (0.5 mg/100 g body wt.) within 2 h. It was found that changes in the rate of incorporation of labeled amino acids into ACTH were not a reflection of general changes in the rate of tissue protein synthesis.