To clarify which brain regions are activated by stress, we used expression of the proto-oncogene, c-fos, as a marker. An increased number of neurons expressing Fos-like immunoreactivity in their nuclei was observed in discrete brain regions, such as the lateral septum, midline nuclei of the thalamus, paraventricular hypothalamic nucleus, brain stem catecholaminergic, and serotonergic neurons, in response to pain or immobilization stress. Distribution patterns of Fos-like immunoreactive neurons were quite similar in animals subjected to pain or immobilization. Whether or not neurons projecting to the spinal cord to activate the sympathetic pathway express Fos-like immunoreactivity was examined by means of fluorescent double-labeling using fluoro-gold (FG) as a tracer. In the PVH, Fos-immunoreactive neurons were localized in the dorsal medial parvocellular part, although those projecting to the spinal cord were localized dorsally and ventrally. Less than 1% of Fos-positive neurons in this nucleus was colocalized with FG. Among fos-like immunoreactive catecholaminergic and serotonergic neurons in the brain stem, those in the A5 region and raphe pallidus projected to the spinal cord.