Kisspeptin, neurokinin B (NKB) and dynorphin A (Dyn) are coexpressed within KNDy neurons that project from the hypothalamic arcuate nucleus (ARC) to GnRH neurons and numerous other hypothalamic targets. Each of the KNDy neuropeptides has been implicated in regulating pulsatile GnRH/LH secretion. In isolation, kisspeptin is generally known to stimulate, and Dyn to inhibit LH secretion. However, the NKB analog, senktide, has variously been reported to inhibit, stimulate or have no effect on LH secretion. In prepubertal mice, rats and monkeys, senktide stimulates LH secretion. Furthermore, in the monkey this effect is dependent on kisspeptin signaling through its receptor, GPR54. The present study tested the hypotheses that the stimulatory effects of NKB on LH secretion in intact rats are mediated by kisspeptin/GPR54 signaling and are independent of a Dyn tone. To test this, ovarian-intact prepubertal rats were subjected to frequent automated blood sampling before and after intracerebroventricular injections of KNDy neuropeptide analogs. Senktide robustly induced single LH pulses, while neither the GPR54 antagonist, Kp-234, nor the Dyn agonist and antagonist (U50488 and nor-BNI, respectively) had an effect on basal LH levels. However, Kp-234 potently blocked the senktide-induced LH pulses. Modulation of the Dyn tone by U50488 or nor-BNI did not affect the senktide-induced LH pulses. These data demonstrate that the stimulatory effect of NKB on LH secretion in intact female rats is dependent upon kisspeptin/GPR54 signaling, but not on Dyn signaling.