Gambel's white-crowned sparrow is a long distance migrant that undergoes spontaneous gonadal regression as a result of long day exposure. This termination of breeding is caused by the development of photorefractoriness and the birds become insensitive to long days, including continuous light. The present study investigated its possible mechanisms by examining the activity of the gonadotrophin-releasing hormone (GnRH) system under different photoperiodic regimes. We investigated the localisation and distribution of GnRH-I, its precursor pro-GnRH-GAP and GnRH-II in Gambel's white-crowned sparrow brain using immunocytochemistry with specific antibodies during photostimulation and the development of photorefractoriness. The study revealed that photoperiodic treatment, including the onset of photorefractoriness, had no significant effect on the size or number of GnRH-I, pro-GnRH-GAP or GnRH II immunoreactive cells, or the density of the GnRH-I, pro-GnRH-GAP immunoreactive fibres at the median eminence. GnRH-II was not found in the median eminence, suggesting that it does not regulate pituitary gonadotrophin secretion. GnRH-I measurement in hypothalamic extracts by radioimmunoassay did not reveal any significant difference between birds that were photostimulated or in the early stages of photorefractoriness. Furthermore, the action of the excitatory amino acid glutamate agonist N-methyl-D-aspartate on GnRH neurones in photorefractory birds was demonstrated by the significant blockade of luteinising hormone release with a specific GnRH antagonist. Taken together, these results suggest that, in Gambel's white-crowned sparrow, a decrease in GnRH-I secretion is the initial step for the onset of photorefractoriness and not a decrease in GnRH-I biosynthesis.