An in vitro bioassay method using Leydig cells from mouse testes has been validated for quantitating luteinizing hormone (LH) in the plasma and cerebrospinal fluid (CSF) of normally menstruating and ovariectomized rhesus monkeys. In all six normally menstruating monkeys studied, the mid-cycle peak of plasma LH coincided with the estradiol peak in blood samples taken at 24-hour intervals. Blood samples collected at 4-hour intervals during the periovulatory period showed multiple peaks of both bioactive LH and estradiol, and the major estradiol peak preceded the major bioactive LH peak by about 16 h. The plasma progesterone levels were about 1 ng/ml at the time of LH peak. Bioactive LH was detected also in the CSF samples of both intact and ovariectomized monkeys. The levels of LH in CSF were not elevated in the gonadectomized monkeys although their plasma LH levels were markedly increased.