Cholecystokinin (CCK) and substance P (SP) are thought to play an important role in a variety of stress responses. Both CCK- and SP-positive fibers innervating the thalamus are found principally in the midline nuclei, including the paraventricular thalamic nucleus (PVT), which has strong reciprocal connections with the medial prefrontal cortex. In the present study, we determined the source of the CCK- and SP-immunoreactive fibers to the PVT, employing combination of retrograde neuronal tracing and immunohistochemistry in the rat. The PVT-projecting neurons showing CCK immunoreactivity were detected in the dorsomedial nucleus of the hypothalamus, and ventral mesencephalic periaqueductal gray, including the Edinger-Westphal nucleus and the dorsal raphe nucleus. Sources of SP afferents to the PVT were detected in the Edinger-Westphal nucleus, the mesopontine tegmentum and the medullary raphe nucleus. CCK- and SP-immunoreactive fibers may exert modulatory influence on the prefrontal cortical activity via the PVT and regulate behavioral components of stress-adaptation responses.