A double antibody immunoprecipitation technique using affinity-purified adrenocorticotropic hormone (ACTH) antiserum was employed to investigate the biosynthesis of ACTH in a mouse pituitary tumor cell line. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of cell extracts resolved four forms of ACTH with apparent molecular weights of 4,500, 13,000, 23,000, and 31,000. These four forms of ACTH can be detected by radioimmunoassay of cell extracts or by immunoprecipitation of cell extracts following incubation of cultures in [3H] tryptophan, [3H] lysine, or [3H] tyrosine. The double antibody immunoprecipitation scheme developed is specific, quantitative, and reproducible. ACTH biosynthesis was examined in both steady and pulse-labeling experiments using [8H] tyrosine or [3H] lysine. The results of these experiments are consistent with the proposal that Mr=31,000 ACTH is the biosynthetic precursor for all three smaller forms of ACTH and that Mr=23,000 ACTH is a biosynthetic intermediate. Both Mr=13,000 ACTH and Mr=4,500 ACTH are derived from the intracellular processing of Mr=31,000 ACTH.