Several lines of evidence support a role for pituitary adenylate cyclase-activating polypeptide (PACAP) in the regulation of energy balance. In the present study, we have used fluorescent in situ hybridization and immunohistochemistry to investigate in detail the cellular localization and chemical content of PACAP mRNA- and peptide-containing neuronal cell bodies in the mediobasal hypothalamus of the rat. PACAP mRNA-containing cell bodies were demonstrated in high numbers in the ventromedial hypothalamic nucleus (VMH) and in lower numbers in the arcuate nucleus (Arc). In colchicine-treated rats, PACAP immunoreactivity was demonstrated in many cell bodies of the VMH and several cell bodies of the ARC. Double-labeling revealed that PACAP immunoreactivity was present in approximately 20% of pro-opiomelanocortin (POMC) neurons in the ventrolateral Arc as shown by presence of alpha-melanocyte-stimulating hormone (alpha-MSH), but not in agouti-related peptide (AgRP)-containing neurons in the ventromedial aspect of the Arc. PACAP immunoreactivity was also colocalized with the vesicular acetylcholine transporter (VAChT; a marker for cholinergic neurons) in Arc POMC neurons. Brainstem POMC neurons in the commissural part of the solitary tract nucleus were devoid of PACAP immunoreactivity. However, several VAChT-positive neurons in the dorsal motor nucleus of the vagus nerve were also PACAP immunoreactive, whereas VAChT-positive neurons of the motor nucleus of the hypoglossal nerve were PACAP-negative. The results show presence of PACAP with alpha-MSH in a subpopulation of hypothalamic POMC neurons and point further to the neurochemical heterogeneity of hypothalamic, but not brainstem, POMC neurons.