Following bilateral adrenalectomy in adult male rats, there occurs a pattern of triphasic change in basal plasma concentration of radioimmunoassayable ACTH. Plasma ACTH is markedly elevated at 2 h, has returned down almost to normal at 20 h and is again markedly elevated 96 h after adrenalectomy. We have examined serotonin (5HT) synthesis rates in several brain regions, anterior hypothalamus, posterior hypothalamus, and brain stem, at these times after adrenalectomy using the accumulation of 5-hydroxytryptophan (5HTP) after inhibition of aromatic L-amino acid decarboxylase with m-hydroxybenzylhydrazine. In both anterior hypothalamus and brain stem, decreased 5HT synthesis rates were observed at 2 and 96 h after adrenalectomy, but at 20 h 5HT synthesis rates were normal. This pattern was not observed in the posterior hypothalamus. Thus, we demonstrated inverse correlations between 5HT synthesis rates in anterior hypothalamus and brain stem, but not posterior hypothalamus, and basal plasma ACTH concentration throughout the period of triphasic change following adrenalectomy in adult male rats. Both the adrenalectomy-induced increases in plasma ACTH concentration and the adrenalectomy-induced decreases in brain 5HT synthesis rates were inhibited by treatment with dexa-methasone, suggesting that the changes resulted from glucocorticoid withdrawal. The data are consistent with a role of brain 5HT neurons with cell bodies in brain stem and nerve endings in anterior hypothalamus in the regulation of the triphasic changes in plasma ACTH concentration following adrenalectomy in rats.