Seasonally breeding animals undergo numerous physiological changes in response to changes in the length of the photoperiod. In most warm-weather breeding rodents, these changes result in reproductive quiescence during short photoperiods. It has been hypothesized that this change is mediated by changes in the activity of gonadotropin-releasing (GnRH) hormone neurons of the hypothalamus. This study was designed to test whether there are changes in the releasable pool of GnRH in the hypothalamus in response to changes in photoperiod, the presence of gonadal steroids, or the responsiveness of the individual animal to photoperiodic changes. Male deer mice (Peromyscus maniculatus) were maintained on long or short day photoperiod and either left intact, castrated, or castrated with testosterone replacement. KCl-evoked GnRH release was measured from hypothalamic explants from each animal and compared between long and short days, between castrated, intact, and castrated with testosterone replacement animals, and between animals that did or did not show gonadal regression in response to short day treatment. There was a significant decline in evoked release of GnRH in short day housed animals when comparing photoperiod responsive mice to nonresponsive mice. In addition, both reproductively nonresponsive and long day-housed mice release less GnRH following castration than their intact counterparts. When castrated long day-housed mice were provided with long day levels of testosterone, evoked GnRH release was restored to intact levels. Taken together, the results of this study suggest that variation in testicular response to short days is most likely due to differences in the release of GnRH from the hypothalamus.