Kisspeptin neurons are indispensable for reproduction. Here we present the generation of a Kiss-Cre knock-out model, with a Cre-recombinase (Cre) gene cassette inserted at the Kiss1 gene ATG initiation codon. The endogenous Kiss1 promoter directs expression of Cre specifically in kisspeptin neurons. Female and male mice with the Cre insertion at both alleles failed to undergo pubertal sexual maturation due to disrupted Kiss1 transcription. When crossed to a Rosa26-CAG-tdTomato reporter mouse, we are able to visualize the three-dimensional neuroanatomical distribution of Kiss1-expressing neurons within the hypothalamus using the newly developed CLARITY method. The fluorescent tomato expression pattern in the Kiss1-Cre;R26tdTomato/+adult mouse recapitulates what has been reported earlier for kisspeptin neurons in mouse brain, with prominent expression in the arcuate nucleus (ARN; both males and females) and the rostral periventricular area of the third ventricle (RP3V) for females. We observed the tomato-expressing fibres from RP3V project to the rostral preoptic area in adult females. The fibres also project to the lateral hypothalamus and extend caudally to the ARN. Immunofluorescence was performed to determine the co-labeling of tomato-expressing cells with kisspeptin to identify the constitutive Kiss1-expressing cells. The percentage of co-labeling in the rostral ARN of gonadectomized Kiss1-Cre; R26tdTomato/+ mice is 78.5 ±3.9% for females (n=5) and 76.8 ±5.0% for males (n=4). In intact females (n=5), 65.2 ±5.8% of tomato-expressing cells in the AVPV and 72.5 ±5.0% within the PVpo are constitutively expressing Kiss1. We are currently using the Kiss1Cre;R26tdTomato/+ male mouse to examine the biophysical properties of ARN kisspeptin neurons. Whole-cell recordings revealed that ARN kisspeptin neurons exhibit irregular spiking activities driven by transient outward A-type potassium currents. This spiking irregularity is similar to that found in rat cortical neurons and is hypothesized to have a role in local neuronal network regulation. Supported by BBSRC grant (BB/FO1936X/1).