The serotonin (5-hydroxytryptamine, 5-HT) precursor 5-hydroxy-L-tryptophan (5-HTP) and 5-HT antagonists, respectively, are able to stimulate and block pituitary adrenocorticotropin (ACTH) release. However, our previous data do not support a role of central serotoninergic systems in the neural control of ACTH release. We thus examined the hypothesis that 5-HTP given either alone or with uptake-blocking drugs such as fluoxetine caused stimulation of ACTH through activation of central noradrenergic neuronal activity (NNA). The hypothesis was tested in normal adult male rats by correlating medial basal hypothalamic NNA and serotoninergic neuronal activity (SNA) with serum ACTH following administration of 5-HTP (20 and 100 mg/kg, i.p.) in the presence or absence of fluoxetine (10 mg/kg, i.p.) or cyproheptadine (10 mg/kg, i.p.). The α2-adrenergic agonist clonidine (150 µg/kg, i.p.) was used to inhibit central NNA and to examine the role of α2-adrenoceptors in the actions of serotoninergic drugs. Computerized mass spectrometry was employed to specifically and precisely assay hypothalamic norepinephrine (NE), 3,4-dihydroxyphenylethyleneglycol (DHPG), 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) to obtain indices of hypothalamic NNA (DHPG/NE) and SNA (5-HIAA/5-HT). The administration of fluoxetine/5-HTP stimulated (p < 0.01) both hypothalamic NNA and ACTH release. Serum ACTH levels significantly correlated with hypothalamic NNA (p < 0.0001) and SNA (p < 0.005), however, multiple linear regression analysis revealed that central NNA (p 0.4) was a significant determinant of ACTH release. Further, clonidine, the inhibitor of NNA, completely blocked (p < 0.01) the ACTH response to fluoxetine/5-HTP. The administration of high-dose 5-HTP resulted in significant stimulation of both hypothalamic NNA and serum ACTH but, in this case, clonidine failed to block 5-HTP-induced ACTH release, despite the inhibition of hypothalamic NNA, and there was no strong relationship between serum ACTH and NNA. However, when cyproheptadine was administered together with 5-HTP there was a small, but significant, suppression of the ACTH response and the strong relationship between serum ACTH and hypothalamic NNA was restored. In the presence of cyproheptadine, clonidine treatment resulted in complete inhibition of the ACTH response to high-dose 5-HTP. Thus, exogenous 5-HTP may stimulate ACTH release via two different mechanisms. The predominant one is mediated centrally via activation of NNA and can be blocked by clonidine but not 5-HT antagonists. The other is due to a direct 5-HT (or 5-HTP) action, apparently not involving hypothalamic monoamine neuronal pathways, and cannot be blocked by clonidi