Progesterone (P) is the primary effector of LH (and by inference gonadotropin-releasing hormone) pulse frequency slowing in cycling women, but the time course of this action is unclear. We hypothesized that P administration to estradiol (E₂)-pretreated women would slow LH pulse frequency within 12 hours. We studied eight normally cycling women in two separate cycles (follicular phase, cycle days 7-11). After 3 days of E₂ pretreatment (0.2 mg/day via transdermal patches), a 25-hour blood sampling protocol (starting at 0800 h) was performed to define LH pulsatility. Oral micronized P (100 mg) or placebo (PBO) was administered at 1800 h in a randomized, double-blind fashion, with treatment crossover occurring during a subsequent cycle. The 10-hour mean P concentration increased from 0.6 ± 0.1 ng/ml before P (0800-1800 h) to 3.9 ± 0.3 ng/ml after P administration (2200-0800 h; P < 0.01). Ten-hour mean LH interpulse interval increased significantly after both P and PBO administration, with no significant difference between P and PBO. In contrast, mean LH, LH amplitude, and mean FSH increased significantly within 4 hours of P administration, but not after PBO. We conclude that in E₂-pretreated women in the late follicular phase, (1) nocturnal LH pulse frequency is not acutely (within 12 hours) influenced by P administration; (2) an acute increase in P causes pronounced augmentation of gonadotropin pulse amplitude within 4 hours; and (3) LH pulse frequency slows overnight during the second half of the follicular phase.