Kisspeptin plays a critical role in pubertal timing and reproduction. In rodents, kisspeptin neurones located within the hypothalamic arcuate (ARC) and anteroventral periventricular (AVPV) nuclei are thought to be involved in LH pulse and surge generation respectively. Inactivating mutations in kisspeptin or its receptor result in a lack of puberty and hypogonadotrophic hypogonadism, whereas activating mutations cause precocious puberty. With the use of bilateral micro-injections of recombinant adeno-associated virus encoding kisspeptin antisense (rAAV-Kisspeptin-AS) into the ARC or AVPV of postnatal day (pnd) 10 female rats, we investigated the relative importance of these two kisspeptin populations in the control of (i) pubertal timing, (ii) oestrous cyclicity and (iii) LH surge and LH pulse generation. A 37% knockdown of kisspeptin in the AVPV resulted in significant delay in vaginal opening and first oestrous (vaginal opening; pnd 40.4±0.9 vs 36.8±0.9: rAAV-Kisspeptin-AS vs rAAV-EGFP as control, n=9-12; P<0.05), abnormal oestrous cyclicity and reduction in the occurrence of spontaneous LH surges, though of normal amplitude, but had no effect on LH pulse frequency (the latter measured following ovariectomy). A 34% reduction of kisspeptin in ARC had no effect on puberty onset (vaginal opening; pnd 36.7±0.6 vs 37.1±0.9:rAAV-Kisspeptin-AS vs control, n=9-13; P=0.68), but resulted in abnormal oestrous cyclicity and decreased LH pulse frequency. Additionally, the knockdown of kisspeptin in the ARC decreased the amplitude but not the incidence of spontaneous LH surges. These results suggest a critical importance for AVPV, but not ARC, kisspeptin neurones in the control of pubertal timing. In accordance with our previous studies, ARC kisspeptin signalling is critical for normal pulsatile LH secretion in female rats. However, despite the classical association of AVPV kisspeptin neurones in LH surge generation, the results of this study suggest that both AVPV and ARC populations are essential for normal LH surges and oestrous cyclicity.