The arcuate nucleus gives rise to approximately 80% of the neuropeptide Y (NPY)-immunoreactive (IR) innervation to thyrotropin-releasing hormone (TRH) neurons in the hypothalamic paraventricular nucleus (PVN). However, the source of the remaining 20% is unknown. Since medullary adrenergic neurons synthesize NPY and also innervate the hypophysiotropic TRH neurons, we raised the possibility that adrenergic neurons contribute to the NPY-ergic innervation of TRH neurons in the PVN. Triple-labeling immunofluorescence was performed to study the colocalization of NPY and phenylethanolamine N-methyltransferase (PNMT)--the key enzyme of adrenaline synthesis--in axons in association with hypophysiotropic TRH neurons. NPY-immunoreactivity was observed in 74% of PNMT-IR axon varicosities apposed to proTRH-IR neurons, comprising 26% of all NPY-IR axons in contact with proTRH perikarya and dendrites in the PVN. We conclude that at least two distinct populations of NPY neurons innervate hypophysiotropic TRH neurons, the NPY neurons of the hypothalamic arcuate nucleus and the medullary adrenergic neurons that co-contain NPY.