In order to determine whether pituitary prolactin release was directly related to the secretion of TRH into hypophysial portal blood, serial portal and jugular venous blood samples were collected from seven lactating and three non-lactating ewes. In another experiment, samples were collected from five ovariectomized ewes while being exposed to an audio-visual stress and then later administered with chlorpromazine. Secretion of TRH was pulsatile in all ewes and independent of prolactin secretion; TRH pulses coincided with significant increases in prolactin secretion in only 15% of cases and only 29% of prolactin pulses were associated with TRH pulses. Sixty-seven per cent of suckling bouts used were associated with increases in prolactin secretion, but only 22% of these were associated with significant increases in TRH secretion. Chlorpromazine increased prolactin levels fourfold but did not affect portal concentrations of TRH. Audio-visual stress was not a reliable method of causing prolactin release in this model. Mean portal concentrations of TRH and jugular concentrations of prolactin were not significantly correlated. These results show that hypothalamic TRH and pituitary prolactin are secreted independently in the sheep, implying that increases in prolactin release caused by suckling or chlorpromazine are not in the direct result of increased TRH secretion.