Cocaine- and amphetamine-regulated transcript (CART) has stimulatory effects on the hypothalamic-pituitary-adrenal axis through direct effects on hypophysiotropic CRH neurons. Recently, CART-containing axons have been demonstrated to densely innervate the hypophysiotropic CRH neurons. Based on the sources of the CART-immunoreactive (IR) innervation of the PVN, the putative origin of these CART-containing fibers include neurons of the hypothalamic arcuate nucleus that co-express -melanocyte-stimulating hormone (-MSH) and medullary adrenaline producing neurons. To determine whether these cell groups contribute to the CART innervation of the hypophysiotropic CRH neurons, we performed quadruple-labeling immunofluorescent study using antisera against CRH, CART, -MSH and phenylethanolamine-N-methyl-transferase (PNMT), the latter as a marker for adrenaline. Consistent with previous observations, PNMT- and CART-IR axons densely innervated all CRH neurons, whereas the -MSH-IR innervation was sparse. While approximately 60% of CART-IR varicosities in juxtaposition to CRH neurons co-contained PNMT, only approximately 18% of them were immunopositive for -MSH. All -MSH-IR boutons and approximately 90% of PNMT-containing varicosities on the surface of CRH neurons were also labeled for CART. The remaining 22% of CART axon varicosities in contact with CRH neurons contained neither -MSH or PNMT. These results indicate that medullary adrenergic/CART neurons are the major source for the CART innervation of CRH neurons in the PVN, however, to a lesser extent the arcuate nucleus also contributes to the CART-IR innervation of these neurons. The observation that nearly 20% of the CART-IR afferents contain neither -MSH nor PNMT, however, suggests that additional sources also contribute to the CART-IR input of hypophysiotropic CRH neurons.