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A radioimmunoassay for plasma ACTH has been described and evaluated. Rabbit antiserum produced by immunization with Asp²⁵, Ala²⁶, Gly²⁷,]-_h-corticotrophin-(1–28)-octacosa-peptide (a sequence analogue of _h^1-28-ACTH) bovine gamma globulin conjugate was used. The antiserum is specific for the NH₂-terminal portion of the ACTH molecule and cross-reactivity of human, porcine and rat ACTH in the system has been demonstrated. Reasonable agreement was found between estimates obtained by bioassay and radioimmunoassay of the ACTH content of rat pituitary gland incubation media, indicating a close relationship between the sequence of ACTH recognized by the antibodies and the sequence possessing the steroidogenic activity. Measurement of the amount of ACTH in the plasma required the preliminary extraction and concentration of the hormone. Over a range of concentrations between 3·5 and 3600 pg/ml, extraction recovery was independent of the initial concentration of ACTH in the plasma. Extraction gave rise to no changes in the immunological properties of standard ACTH. The concentration of immunoreactive ACTH in rat plasma was 48 ± 3·6 (S.E.M.) pg/ml in the morning and 106 ± 9·9 pg/ml in the afternoon. Exposure to ether for 5 min and subsequent laparotomy gave rise to a significant increase in the concentration of immunoreactive ACTH in the plasma. The resting level of ACTH and the ACTH response to stress were both significantly higher 1 and 7 days after adrenalectomy. Intravenous injection of a hypothalamic extract elicited a considerable rise in the concentration of immunoreactive ACTH in the plasma, but no response was seen after oral administration of this partially purified extract. The sensitivity, precision and specificity of this ACTH radioimmunoassay make it a useful tool for studying pituitary– adrenal physiology.