We tested the hypothesis that kisspeptin cells constitute the pulse generator for GnRH secretion. In ewes, we determined whether iv administered kisspeptin elicits a secretory pulse of LH in anaesthetized, sex-steroid suppressed ovariectomized ewes. A response was seen in both anaesthetized and conscious animals, which was not associated with induction of c-Fos labeling in GnRH cells, supporting the notion that kisspeptin acts on the neurosecretory GnRH terminals. Response was lower in the anaesthetized animals, suggesting that some nonkisspeptin elements may be involved in GnRH responses. Microinjection of kisspeptin (100 nmol) into the median eminence of conscious ewes elicited a pulse of LH, indicating that kisspeptin acts at this level to cause GnRH secretion. To determine which cells are activated at the time of GnRH secretion, we blood sampled 18 ewes during the luteal phase of the estrous cycle and harvested brains after 3 hours. Three of these ewes displayed a pulse of LH within 30 minutes of euthanasia. An increase in c-Fos labeling was seen in kisspeptin and glutamate cells of the arcuate nucleus but not in GnRH neurons, preoptic kisspeptin neurons, or preoptic glutamate neurons. Immunohistochemistry in 4 hypothalami showed that 72 of arcuate kisspeptin cells receive glutamatergic input. These data support the concept that the kisspeptin cells of the arcuate nucleus drive pulsatile secretion of GnRH at the level of the median eminence, but this may involve upstream input from glutamate cells. We conclude that the pulse generator for GnRH secretion involves more than 1 element.