Kisspeptin-Gpr54 signaling is critical for the GnRH neuronal network controlling fertility. The present study reports on a kisspeptin (Kiss)-green fluorescent protein (GFP) mouse model enabling brain slice electrophysiological recordings to be made from Kiss neurons in the arcuate nucleus (ARN) and rostral periventricular region of the third ventricle (RP3V). Using dual immunofluorescence, approximately 90% of GFP cells in the RP3V of females, and ARN in both sexes, are shown to be authentic Kiss-synthesizing neurons in adult mice. Cell-attached recordings of ARN Kiss-GFP cells revealed a marked sex difference in their mean firing rates; 90% of Kiss-GFP cells in males exhibited slow irregular firing (0.17 ± 0.04 Hz) whereas neurons from diestrous (0.01 ± 0.01 Hz) and ovariectomized (0 Hz) mice were mostly or completely silent. In contrast, RP3V Kiss-GFP cells were all spontaneously active, exhibiting tonic, irregular, and bursting firing patterns. Mean firing rates were significantly (P < 0.05) higher in diestrus (2.1 ± 0.3 Hz) compared with ovariectomized (1.0 ± 0.2 Hz) mice without any changes in firing pattern. Recordings from RP3V Kiss-GFP neurons at the time of the proestrous GnRH surge revealed a significant decline in firing rate after the surge. Together, these observations demonstrate unexpected sex differences in the electrical activity of ARN Kiss neurons and markedly different patterns of firing by Kiss neurons in the ARN and RP3V. Although data supported a positive influence of gonadal steroids on RP3V Kiss neuron firing, no direct evidence was found to support the previously postulated role of ARN Kiss neurons in the estrogen-negative feedback mechanism.