The anterior parvocellular subdivision of the PVN (aPVN) contains nonhypophysiotropic thyrotropin-releasing hormone (TRH) neurons that are densely innervated by feeding-related neuronal groups of the hypothalamic arcuate nucleus. To determine how these TRH neurons are integrated within the brain, the major projection fields of this cell group were studied by anterograde and retrograde tract-tracing methods. Projection sites were identified by injection of the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHAL) into the aPVN, and subsequent double immunofluorescent staining was used to visualize axons containing both PHAL and pro-TRH. To distinguish between the projection sites of TRH neurons residing in the aPVN and the closely situated perifornical area, the retrograde tracer cholera toxin B subunit (CTB) was injected into regions where PHAL/pro-TRH-containing axons were densely accumulated. TRH neurons in the aPVN were found to project to the hypothalamic arcuate, dorsomedial and ventral premammillary nuclei, medial preoptic region, tuber cinereum area, paraventricular thalamic nucleus, bed nucleus of the stria terminalis, lateral septal nucleus, and central amygdaloid nucleus. Projection fields of perifornical TRH neurons were in partial overlap with those of the aPVN TRH cells. In addition, these neurons also innervated the hypothalamic ventromedial nucleus, the medial amygdaloid nucleus, and the amygdalohippocampal area. The data suggest that, through its efferent connections, aPVN TRH neurons may be involved in the regulation of energy homeostasis coordinately with effects on behavior, locomotor activity, and thermogenesis. In addition, the major differences in the projection fields of aPVN and perifornical TRH neurons suggest that these two TRH-synthesizing neuronal groups are functionally different. Copyright 2009 Wiley-Liss, Inc.