Kisspeptins are small peptides encoded by the Kiss1 gene that have been the focus of intense neuroendocrine research during the last decade. Kisspeptin is now considered to have important roles in the regulation of puberty onset and adult oestrogen-dependent feedback mechanisms on gonadotrophin-releasing hormone secretion. Several kisspeptin antibodies have been generated that have enabled an overall view of kisspeptin peptide distribution in the brain of many mammalian species. However, it remains that the distribution of the different kisspeptin isoforms is unclear in the mammalian brain. In the present study, we report on two new N-terminal-directed kisspeptin antibodies, one against the mouse kisspeptin-52 sequence (AC053) and one against the rat kisspeptin-52 sequence (AC067), and use them to specifically map these long isoforms in the brains of mouse and rat, respectively. Kisspeptin-52 immunoreactivity was detected in the two main kisspeptin neuronal populations of the rostral periventricular area and arcuate nucleus but not in the dorsomedial hypothahamus. A large number of fibres throughout the ventral forebrain were also labelled with these two antibodies. Finally, a comparison with the most commonly used C-terminal-directed kisspeptin antibodies further suggests the presence of shorter kisspeptin fragments in the brain with specific inter- and intracellular expression patterns.