In the past decade gonadotrophin inhibitory hormone (GnIH) has been found to regulate reproductive activity at the level of the hypothalamus through its actions on GnRH. We investigated the role of GnIH in the mare, a long day breeder, which experiences a well characterised transition period between the non-cycling winter period and the breeding season. In this study we collected perfusion-fixed hypothalami from mares (n=3/group) in the three distinct seasonal stages (winter anoestrus, vernal transition and summer cyclicity), and at three points during the oestrous cycle; preovulatory, early luteal and late luteal phase. We used fluorescent immunohistochemistry to determine the hypothalamic locations of GnIH neurons, and to see if seasonal or cycle stages affect the neuronal populations. GnIH immunoreactive (-ir) neurons were found mainly in the dorsomedial nucleus, but populations were also identified in the pre optic area, ventromedial nucleus and in the periventricular region. Cell numbers were higher in the caudal and middle part of the hypothalamus compared with rostral hypothalamus and preoptic area, where few cells were observed. GnIH-ir fibres surrounded the third ventricle along the rostro-caudal extent of the hypothalamus, and extended into the median eminence. Among the cycling animals, GnIH-ir cell numbers were much lower during the early luteal phase (P<0.05) than in the other stages. In the seasonal study, numbers of GnIH-ir neurons were highest (P<0.05) in seasonally anoestrous mares, lower during seasonal transition and lowest in cycling mares (early luteal). The data is in line with an inhibitory action for GnIH in seasonality of reproduction, but the data from cycling animals, where GnIH cell numbers are low at a time when GnRH/LH secretion is low appears counterintuitive. Nonetheless these data suggest a role for GnIH in regulation of oestrous cyclicity and in seasonal reproduction.