Pro-opiomelanocortin (POMC) and agouti-related protein (AGRP) neurons in the hypothalamus regulate various aspects of energy homeostasis and metabolism. POMC and AGRP neurons, respectively, agonize and antagonize melanocortin receptors on their common downstream neurons. However, it is unknown whether they also reciprocally stimulate and inhibit the same neurons by amino acid transmitters. Whereas AGRP neurons are mostly GABAergic, surprisingly, only a small population of POMC neurons has been found to be glutamatergic, and a significantly larger subpopulation to be GABAergic. To further examine amino acid phenotypes of POMC neurons, we studied mRNA expression for the glutamatergic marker, type 2 vesicular glutamate transporter (VGLUT2), and the GABA synthetic enzyme, glutamic acid decarboxylase 67 (GAD67), in POMC neurons of both rats and mice by using in situ hybridization techniques. In rats, approximately 58% of POMC neurons were labeled for VGLUT2 and 37% for GAD67 mRNA. In mice, approximately 43% of POMC neurons contained VGLUT2, and 54% contained GAD67 mRNA. In both species, a prominent mediolateral distribution pattern was observed at rostral and mid levels of the POMC cell group with VGLUT2–POMC neurons dominating in lateral portions and GAD67–POMC neurons in medial portions. These data demonstrate that both glutamatergic and GABAergic cells are present in comparably significant numbers among POMC neurons. Their glutamatergic or GABAergic phenotype may represent a major functional division within the POMC cell group. J. Comp. Neurol. 521:3287–3302, 2013. © 2013 Wiley Periodicals, Inc.