Hypophysiotropic thyrotropin-releasing hormone (TRH)-synthesizing neurons, the central regulators of the hypothalamic-pituitary-thyroid axis, are located in the paraventricular nucleus of the hypothalamus (PVN). These neurons are well-known to be stimulated by cold exposure through activation of ascending brainstem pathways, and are heavily innervated by catecholaminergic axons that contain dopamine-beta-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT), enzymes that generate noradrenaline and adrenaline, respectively. However, whether noradrenergic-synthesizing cell groups that lack PNMT contribute to the innervation of TRH neurons is not known. Therefore, triple-labeling immunofluorescence was performed using antibodies against DBH, PNMT and proTRH to determine the relative involvement of adrenaline-synthesizing and noradrenergic neurons in the innervation of TRH neurons in the PVN of rats. Using confocal microscopy, the number of PNMT/DBH (adrenaline-synthesizing) and single-labeled DBH (noradrenergic) boutons juxtaposed to proTRH neurons was quantified. Both noradrenergic and PNMT-containing varicosities were observed in close apposition to virtually all proTRH neurons. An average of 11.8±0.6 PNMT-containing and 7.4±1.0 noradrenergic boutons was present on the surface of proTRH cell bodies and proximal dendrites. Of all catecholaminergic axon-varicosities juxtaposed to proTRH neurons, 63.5±1.2% contained PNMT while the remaining 36.5±1.2% were immunopositive for DBH only. We conclude that both adrenaline-synthesizing and noradrenergic axons innervate hypophysiotropic TRH neurons, although there is a predominance of adrenaline-synthesizing fibers. Since adrenaline-synthesizing and noradrenergic cell groups of the brainstem may respond differently to various physiological stimuli, we hypothesize that the two cell groups are likely to mediate the effects of distinct stimuli toward the hypophysiotropic TRH neurons.