The hyposecretion of growth hormone (GH) in maternal separation (MS) of rat pups is remarkably similar to the specific suppression of GH secretion to evocative tests in infants diagnosed with Reactive Attachment Disorder of Infancy (RADI). Growth hormone-releasing factor (GRF) and somatostatin (SS) provide opposing regulation of GH secretion, and both are modified by noradrenergic and serotonergic stimuli in neonatal and adult rats. In this study, GRF administration reversed MS-induced suppression of GH secretion in 10-day-old pups, but this action of GRF was prevented by pretreatment with cyproheptadine (Cypro), a serotonergic antagonist. The normalization of GH secretion after return to the dam was not altered by pretreatment with SS. Indirect 5-HT agonists, fluoxetine (FLX) and 5-HTP, both stimulated GH secretion in 10-day-old pups. All mixed serotonin- and 5-HT1A-receptor agonists suppressed GH secretion in 10-day-old pups. Antagonists Cypro and ketanserin (Ket) suppressed FLX-induced GH secretion. In contrast, only Cypro suppressed 5-HTP-induced GH secretion. Maternal separation inhibited GH secretion stimulated by 5-HTP, but not by FLX. The serotonergic pathway acting on 5-HT2A receptors may be obligatory for GRF-mediated stimulation and is sensitive to inhibition by Cypro. In addition, a Ket-sensitive serotonergic parallel pathway acting on 5-HT2C receptors may also stimulate GH secretion by acting on GRF or SS. However, only the obligate 5-HT2A pathway appears to be suppressed in MS. These data and observations by others indicate that specific suppression of GH secretion in MS may derive from a reduction in GRF release through noradrenergic neurons, possibly impinging upon serotonergic terminals in the hypothalamus. This study may also provide insight into mechanisms by which GH secretion is suppressed in humans with RADI.