TRH and CRH are secreted into the hypophysial portal circulation by hypophysiotropic neurons located in parvicellular subdivisions of the hypothalamic paraventricular nucleus (PVH). Recently these anatomical compartments of the PVH have been shown to contain large numbers of glutamatergic neurons expressing type 2 vesicular glutamate transporter (VGLUT2). In this report we presented dual-label in situ hybridization evidence that the majority (>90%) of TRH and CRH neurons in the PVH of the adult male rat express the mRNA encoding VGLUT2. Dual-label immunofluorescent studies followed by confocal laser microscopic analysis of the median eminence also demonstrated the occurrence of VGLUT2 immunoreactivity within TRH and CRH axon varicosities, suggesting terminal glutamate release from these neuroendocrine systems. These data together indicate that the hypophysiotropic TRH and CRH neurons possess glutamatergic characteristics. Future studies will need to address the physiological significance of the endogenous glutamate content in these neurosecretory systems in the neuroendocrine regulation of thyroid and adrenal functions.