To study the locus at which opioids act to release PRL in vivo, beta-endorphin (beta-EP) was injected into intact and pituitary stalk-sectioned monkeys. In each of five intact monkeys, serum PRL rose to peak concentrations of 200-300% of baseline 20 min after injection. In contrast, beta-EP failed to cause any PRL increase in four stalk-sectioned animals. Beta-EP also failed to stimulate PRL in two stalk-sectioned monkeys receiving estrogen replacement, indicating that estrogen deficiency was not the cause of their failure to respond. To test possible antagonism of dopamine by beta-EP directly at the pituitary, L-dopa was given to six stalk-sectioned monkeys with and without beta-EP pretreatment. No alteration of the PRL suppression by L-dopa was observed Disappearance of injected beta-EP from plasma was studied in four intact monkeys. Initial and terminal half-lives ranged from 2.3-4.0 min and from 16.0-30.2 min, respectively; MCRs ranged from 70-170 ml/min. We conclude that beta-EP does not stimulate PRL secretion either directly or by interacting with dopamine at the pituitary level. These results support a hypothalamic rather than a direct pituitary site of action for opioid-stimulated PRL release.