The sheep exhibits a marked sex difference in the timing of the pubertal increase in luteinizing hormone (LH). Male lambs undergo a reduction in sensitivity to inhibitory steroid feedback, leading to an increase in LH by 10 weeks of age, but females remain hypersensitive until 30 weeks of age. Endogenous opioids suppress LH secretion in the female lamb prepubertally and in adult male and female sheep. It has been suggested that a reduction in opioid inhibition of LH secretion is the signal to time puberty. Therefore, if a decrease in opioid tone occurs during sexual maturation, it should begin earlier in the male lamb than in the female. The objective of this study was to compare opioid inhibition of LH secretion in male and female lambs in relation to the timing of puberty. Our approach was to examine the response to the opioid antagonist naloxone at various ages in both sexes. To determine the timing of the pubertal LH rise in the presence of constant inhibitory steroid feedback, male and female lambs (n = 5 each) were gonadectomized at 3 weeks of age and implanted with a Silastic capsule of estradiol. They were then challenged with naloxone at 5, 11, and 23 weeks of age; blood samples were collected every 12 min for 8 hours, and lambs received naloxone (1 mg/kg i.v.) at hours 4, 5, 6, and 7. Mean LH before and during naloxone treatment was compared at each age. During the 4-hour pretreatment period, no LH pulses were evident in prepubertal females at 5, 11, or 23 weeks of age, and mean LH remained low (0.8 ± 0.05 ng/ml). In prepubertal males, mean LH was 1.5 ± 0.2 ng/ml at 5 weeks of age and rose at puberty to 6.8 ± 0.3 ng/ml by 23 weeks of age. Despite this sex difference in the timing of the pubertal LH rise, neither male nor female lambs increased LH secretion in response to naloxone until 23 wseeks of age. At this time, mean LH in females increased by 3.2 ± 0.7 ng/ml over pretreatment values in response to naloxone and increased by 5.0 ± 0.9 ng/ml in males. Thus, although opioid tone is expressed prepubertally in the female lamb, it is not evident until after puberty in the male. In a second experiment, we determined if increasing the concentration of estradiol in circulation affects the gonadotropin response to naloxone. LH pulse frequency during naloxone treatment did not differ in ovariectomized prepubertal females bearing 3-, 10-, or 30-mm implants of estradiol, although the larger implants of estradiol suppressed LH during the pretreatment period. These data suggest that increasing steroid negative feedback does not increase opioid tone, thereby reducing the gonadotropin response to a single dose of naloxone. Together, these studies provide evidence that in sheep the timing of the pubertal rise in LH secretion is not causally related to changes in opioid inhibition; rather, other factors including age or season may influence when endogenous opioids begin to serve a major role in modulating gonadotropin secretion in this species.