The concentration of biologically active LH in rhesus monkey (Macaca mulatta) serum was measured by a highly sensitive bioassay based upon testosterone production by dispersed rat interstitial cells. The sensitivity of the in vitro bioassay was equal to or higher than that of radioimmunoassay, with detection limits of 0.1 mIU of human menopausal gonadotropin (hMG) or 10 ng of a rhesus pituitary gonadotropin preparation (LER-1909-2). Parallel dose-response curves were obtained for hMG and rhesus monkey pituitary gonadotropin. The method permits bioassy of LH in 20-100 micronl of serum from adult male monkeys, and from female monkeys during the follicular and luteal phases of the menstrual cycle. Bioactive LH concentrations could be assayed in 0.25 to 5 micronl of serum from mid-cycle, postmenopausal, and castrated female monkeys. Serum LH was undetectable in two hypophysectomized adult female monkeys and six intact immature animals, and was 13+/-6 (SD) mIU/ml in adult male monkeys. In adult females, follicular phase LH levels ranged from 17 to 169 mIU/ml, with a mean of 76+/-52 mIU/ml. The midcycle LH peak was 1738+/-742 mIU/ml and the luteal phase values ranged from 6-47 mIU/ml, with a mean of 35+/-5 mIU/ml. Serum LH concentrations ranged from 100 to 900 mIU/ml in two menopausal females, and from 590-1480 mIU/ml in castrated females. Treatment of castrated female monkeys with estrogen plus progesterone produced an initial two-fold rise in serum LH within 3 days, followed by a gradual decline to one-fourth to one-tenth of the initial levels after 10 days of treatment. Serum LH was suppressed to undetectable levels during the third week, and remained so for the duration of the 60-day treatment period. Bioactive serum LH levels were comparable to levels determined by radioimmunoassay during the follicular and luteal phases of the menstrual cycle, with increased bio-immunoratio at the midcycle peak. The concentrations of biologically active serum LH in rhesus monkeys were similar to those in the human female during the follicular and luteal phases of the menstrual cycle, and were higher at midcycle and after castration. Serum LH levels measured by the interstitial cell bioassay in the rhesus monkey showed appropriate physiological changes and responses to gonadal steroid administration. Furthermore, the bioassay did not detect the LH-like material measured by heterologous radioimmunoassay in the serum of hypophysectomized, immature and steroid-suppressed monkeys. Thus, the rat interstitial cell assay provides a sensitive and valid procedure for measurement of biologically active LH in the serum of these non-human primates.