The possibility that adrenergic receptor antagonists which prevent naloxone-induced release of luteinizing hormone (LH) in vivo exert their action by direct competition with naloxone for hypothalamic opiate receptors was investigated in vitro in immature female rats. First, 26-day-old rats were injected with prazosin, an alpha 1-adrenergic blocker, or yohimbine, an alpha 2-adrenergic blocker, before receiving naloxone (2.5 mg/kg body wt.). Both adrenergic antagonists prevented naloxone-provoked LH secretion in a dose-dependent manner with yohimbine exhibiting a slightly greater potency. In a separate experiment hypothalami from 26-day-old rats were removed, membrane pellets prepared and incubated with [3H]naloxone in the presence of increasing concentrations of naloxone or various monoamine-active substances. Phentolamine, prazosin and yohimbine were the most effective competitors for naloxone binding sites while pronethalol, methysergide and metergoline were far less effective. These findings parallel the relative inhibitory potencies of these compounds in vivo for preventing naloxone-induced LH release as shown here and in a previous report. Clonidine and L-phenylephrine, both alpha-adrenergic agonists, also showed activity in the binding assay. Surprisingly, alpha-methyl-p-tyrosine and 5-hydroxytryptophan, substances which substitute for monoamine precursors early in the biosynthetic pathway, also displaced [3H]naloxone from hypothalamic receptors. These results offer a mechanism for the modulating effects of monoamine-active drugs on opiate antagonist-induced LH release and may have significance for inhibition of LH secretion by endogenous opiates.