Serum LH levels in rhesus monkeys are commonly measured by a radioimmunoassay (RIA) utilizing ovine LH as the radioligand and a novel antiserum to ovine LH. Although meeting most criteria of validity, this RIA cross-reacts with serum from demonstrably hypophysectomized monkeys as well as serum from pre-pubertal monkeys. We have utilized the recently developed rat interstitial cell-testosterone assay (RICT) for measurement of serum LH in rhesus monkeys during various endocrine states and have compared the results with those obtained by RIA. The RICT in vitro bioassay was as sensitive, precise, and accurate as the RIA. Partially purified pituitary monkey LH preparations, varying by as much as 100-fold in purity, had similar potency estimates by both assays. The expected biphasic pattern of serum LH levels during the menstrual cycle as well as the usual increase in serum LH levels after ovariectomy was observed with both assays. In each case, however, basal serum LH levels were overestimated by the RIA and peak serum LH levels were underestimated by the RIA as compared to results obtained by the RICT bioassay. The most striking differences between the two assays were observed with serum from hypophysectomized and prepubertal monkeys. Whereas LH-like activity in such samples was high when measured by the RIA, it was undetectable by the RICT bioassay. The ratios of serum LH (RIA/RICT) varied from greater than 7.2 to greater than 17.4 in such sera. These results demonstrate the serious limitations of the ovine LH RIA for measurement of serum LH in rhesus monkeys. It appears that the RICT bioassay may be a suitable alternative method, especially in circumstances other than the menstrual cycle and after ovariectomy.