Hypothalamic-pituitary stalk portal blood was collected from 12 female rhesus monkeys. The pituitary stalk was approached transorbitally and cut at the level of the diaphragma sellae under direct visualization. After complete heparinization of the animal, stalk portal blood was obtained continuously, for periods of 30 minutes to 9 hours, using a constant exfusion pump at a rate of 30 to 40 mul/min. The mean GnRH in portal blood, as measured by radioimmunoassay, was 66 +/- 6.6 pg/ml (+/- SE) in 7 ovariectomized animals and 51 +/- 5.3 pg/ml (+/- SE) in 2 monkeys during the early follicular phase. Fluctuations in portal blood GnRH were most prominent in ovariectomized animals, with peak levels of 200-800 pg/ml and intervals of 1 to 3 hours between pulses. Peaks of GnRH during the early follicular phase did not exceed 200 pg/ml. The administration of estradiol (1000 ng, iv) to 3 monkeys did not decrease GnRH levels within the next 2 hours. These data provide direct evidence for a hypothalamic mediation of pituitary LH pulsatile release.