Isoforms of the recently cloned vesicular glutamate transporters (VGLUT1-3) selectively accumulate glutamic acid into synaptic vesicles in excitatory axon terminals and are viewed as reliable markers for glutamatergic neurons. Our present studies provided dual-label in situ hybridization evidence that virtually all (99.5%) GnRH neurons express VGLUT2 mRNA in the preoptic region of the adult male rat. Dual-label immunofluorescent experiments were carried out to examine the presence of VGLUT2 protein in GnRH axon terminals. Confocal laser microscopic analysis of the organum vasculosum of the lamina terminalis and the external zone of the median eminence, the major termination fields for GnRH-secreting axons, demonstrated the frequent occurrence of VGLUT2 immunoreactivity in GnRH axon terminals. Together these mRNA hybridization and immunocytochemical data indicate that GnRH neurons of the adult male rat possess marked glutamatergic characteristics. The physiological significance of endogenous glutamate in the regulation of gonadotropin secretion requires clarification.