Abstract The object of this study was to further define the roles of both norepinephrine (NE) and epinephrine (EPIN) in regulating pulsatile luteinizing hormone (LH) release in 4-day ovariectomized rats, in particular to examine the effect of decreasing NE synthesis on pulsatile LH secretion in animals with already greatly depleted levels of brain EPIN. Rats were injected ip with vehicle or drug at -27, -20, -5 and - 3 h relative to the onset of a 3-h blood sampling period. Hypothalamic-preoptic area (HPOA) levels of NE and EPIN were determined by high-performance liquid chromatography. Compared to controls, FLA-63 (25 mg/kg, a dopamine-ss- hydroxylase inhibitor), given at - 3 h, produced 50% and 22% declines in HPOA-NE and EPIN, respectively, and reductions in pulse amplitude and frequency. LY134046 (50 mg/kg, a phenylethanolamine N-methyltransferase inhibitor), given at - 27, - 20 and - 5 h, or -27, -20, -5 and -3 h, produced no change in NE, 88% and 86% declines in EPIN, respectively, and reductions in pulse frequency only. Each LY134046 treatment protocol produced the same decline in EPIN and pulse frequency. Thus, EPIN levels were maximally decreased by three LY134046 injections. When rats were given LY134046 at -27, -20 and -5 h, and FLA-63 at -3 h, compared to rats treated with LY134046 alone, there was no further decrease in HPOA-EPIN (82% decline), a 46% decline in NE, a further reduction in pulse frequency and a reduction in pulse amplitude. This further suppression of LH release must be due to a reduction in HPOA-NE levels since no further decrease in EPIN levels occurred. These data demonstrate within the same animal that NE and EPIN are both stimulatory to pulsatile LH release. NE stimulates the amplitude and frequency, and EPIN stimulates the frequency of pulsatile LH secretion.