The effects of the α<sub>2</sub>-agonist clonidine (CLO), the serotonin (5-HT) precursor 5-hydroxy-L-tryptophan (5-HTP), the 5-HT<sub>2</sub>/histamine (H<sub>1</sub>) antagonist cyproheptadine (CYPRO), the muscarinic cholinergic antagonist atropine (ATR), and an affinity-purified polyclonal anti-rat growth hormone-releasing hormone (rGHRH) immunoglobulin on serum concentrations of growth hormone (GH) and prolactin (PRL) were tested in 2- and 10-day-old litter-mate rat pups. Serum levels of GH and PRL were detected in RIA and Nb2 lymphoma bioassay, respectively. The effects of two different drugs either alone or in combination with each other were evaluated by two-factor analysis of variance. The data indicated that secretion of GH and PRL was regulated by α<sub>2</sub>-adrenergic, serotonergic and cholinergic mechanisms; the pathways regulating the two hormones, however, were distinct. 5-HTP stimulated GH secretion as early as day 2 postpartum via cholinergic mechanisms not involving GHRH; this pathway was also present in 10-day-old pups. An additional serotonergic pathway was functional in 10-day-old pups which mediated CLO-induced release of GH, and did not include cholinergic transmission. The α<sub>2-</sub>adrenergic regulation of GH secretion appeared to involve three distinct mechanisms: (1) a sexually uniform GH-stimulating α<sub>2</sub>-adrenergic pathway was demonstrated with CLO in 2-day-old pups only after pretreatment with ATR; (2) a sexually dimorphic CLO-induced secretion of GH was observed that was mediated by mechanisms sensitive to CYPRO but not to ATR, and occurred by day 10; and (3) 5-HTP-induced GH secretion was counteracted by CLO in 10-day-old pups of both sexes indicating that a sexually uniform GH-inhibiting α<sub>2</sub>-adrenergic pathway was present. The concentration of PRL was not affected by 5-HTP up to day 10, and was decreased by ATR in 10-day-old (but not in 2-day-old) pups. Secretion of GH and PRL appeared to be stimulated by different sets of cholinergic neurons because (1) ATR inhibited GH secretion on day 2 but inhibition of PRL secretion appeared later, and (2) CLO-induced PRL secretion was diminished by ATR, whereas CLO-induced release of GH was not affected in 10-day-old pups. CYPRO increased serum levels of PRL in 10-day-old pups; thus, PRL-inhibiting network was functional at this age. Whether the inhibition of this pathway was due to the serotonin, histamine (H<sub>1</sub>) or dopamine antagonist action of CYPRO requires further investigation.