A radioimmunoassay is described for the measurement of alpha-melanocyte-stimulating hormone (alpha-MSH). The antibody was produced in rabbits by immunization with alpha-MSH coupled to bovine serum albumin with carbodiimide. The antibody did not react significantly with ACTH, beta-MSH, or 6 fragments of ACTH. The sensitivity and reliability of the assay were improved by employing a simple plasma extraction procedure. When applied to a 2 ml plasma sample, the detection limit of the radioimmunoassay was 6 pg/ml. ACTH was measured with a sensitive and specific radioimmunoassay previously described for humans and adapted for the rat. The anti-ACTH serum cross-reacted with the biologically active portion of alpha-p ACTH and not with alpha-MSH, beta-MSH or the alpha-p 17-39 and alpha-p 25-39 fragments of ACTH. The detection limit was 20 pg/ml. Plasma and pituitary alpha-MSH and ACTH had the same immunoreactivity as synthetic alpha-MSH and ACTH. alpha-MSH and ACTH contents of the rat neurointermediate lobe were 1398 +/- 360 (SE) ng and 28.2 +/- 2.9 ng, respectively, while in the anterior lobe they were 102 +/- 31 ng and 551 +/- 36 ng, respectively. The plasma alpha-MSH concentration at 8 AM in male rats was 64 +/- 8 pg/ml when the plasma ACTH concentration was 92 +/- 15 pg/ml. Over a 24-hour period two peaks of plasma alpha-MSH were observed, one at 4 AM (142 +/- 35 pg/ml) and the other at 4 PM (139 +/- 26 pg/ml). Plasma ACTH was higher at noon (151 +/- 43 pg/ml) and 4 PM (130 +/- 48 pg/ml). Short-term exposure to ether induced a transient increase in alpha-MSH level 5 min later and a rapid return to normal levels. Plasma ACTH increased significantly 2.5 min after the onset of ether stress and remained high for 30 min. Two hours' exposure to ether did not change plasma alpha-MSH, although a 3-fold increase in plasma ACTH was observed. Haloperidol injection was followed by a large increase in plasma alpha-MSH, whereas ACTH levels increased similarly after saline and Haloperidol injection. Corticoid administration reduced ACTH, but not alpha-MSH. Three weeks after adrenalectomy, alpha-MSH levels had not changed but ACTH levels had increased ten-fold. These data indicate that alpha-MSH is secreted in the rat, and that the regulation of its secretion is different from that of ACTH.