Gonadotrophin-inhibitory hormone (GnIH) was discovered 8 years ago in birds. Its identification raised the possibility that gonadotrophin-releasing hormone (GnRH) is not the sole hypothalamic neuropeptide that directly influences pituitary gonadotrophin release. Initial studies on GnIH focused on the avian anterior pituitary as comprising the only physiological target of GnIH. There are now several lines of evidence indicating that GnIH directly inhibits pituitary gonadotrophin synthesis and release in birds and mammals. Histological studies on projections from hypothalamic GnIH neurones subsequently implied direct actions of GnIH within the brain and in the periphery. In addition to actions on the pars distalis via the median eminence, GnIH axons and terminals are present in multiple brain areas in birds, and the GnIH receptor is expressed on GnRH-I and -II neurones. Furthermore, we have demonstrated the presence of GnIH and its receptor in avian and mammalian gonads. Thus, GnIH can act directly at multiple levels: within the brain, on the pituitary and in the gonads. In sum, our data indicate that GnIH and its related peptides are important modulators of reproductive function at the level of the GnRH neurone, the gonadotroph and the gonads. Here, we provide an overview of the known levels of GnIH action in birds and mammals. In addition, environmental and physiological factors that are involved in GnIH regulation are reviewed.