The neuropeptide kisspeptin has recently been identified as playing a key role in the activation of gonadotropin-releasing hormone (GnRH) neurons which are the final output cells inducing puberty. The kisspeptin neurons are located in two different regions of the hypothalamus, termed the rostral periventricular area of the third ventricle (RP3V) and arcuate nucleus (ARN). Recent studies show, that these kisspeptin neurons co-express a wide range of different neuropeptides. However, their co-expression of the major amino acid neurotransmitter glutamate and GABA in kisspeptin neurons has received less attention. In the present study, we examined whether GABA and glutamate may also be expressed by RP3V and ARN kisspeptin neurons. To do this we took advantage of new transgenic mouse lines in which all cells expressing the vesicular glutamate transporter 2 (glutamate neurons) or vesicular GABA transporter (GABA neurons) express Cre recombinase. By performing dual labeling immunohistochemistry for Cre (black-stained nuclei) and kisspeptin (brown-stained cytoplasm) we have been able to determine which kisspeptin neurons express GABA or glutamate. Studies were undertaken in adult male and female mice. In males, 65 % of the RP3V and 30 % of ARN kisspeptin neurons were found to co-express GABA. In females only, 25 % of RP3V and Arc kisspeptin neurons were GABAergic. In contrast, the majority of ARN kisspeptin neurons in female mice co-express glutamate (70 %) compared with only 10 % in the RP3V. In males, 35 % of RP3V and 50 % of ARN kisspeptin neurons were glutamatergic. These findings demonstrate that hypothalamic subpopulations of kisspeptin neurons of the RP3V and ARN co-express the neurotransmitter glutamate and GABA in a sexual dimorphic manner. This suggests that the RP3V and ARN kisspeptin neurons are likely to use both GABA and glutamate to control the activity of GnRH neurones.