The central mechanism of stress is poorly understood. This study was designed to examine how corticotropin-releasing factor (CRF) neurons, together with substance P (SP) receptors in the paraventricular hypothalamic nucleus (PVN), central nucleus of the amygdala (CeA), and locus coeruleus (LC), are affected by stress. Sprague-Dawley rats were restrained for 2 h. Animals were sacrificed by decapitation immediately after the 2-h restraint (the 0-h group) and 4, 24, or 48 h after restraint. Tissue sections were cut and collected on two sets of slides. Tissue sections of the first set were processed for studying CRF mRNA using P-33-labeled 60-mer oligonucleotide probe. Immediately adjacent tissue sections were processed for studying SP receptor-binding capacity using I-125-SP ligand. Quantitative results showed that CRF mRNAs in the PVN were significantly up-regulated at the 4- and 24-h stages, and they seemed not to be regulated by SP receptors. In addition, SP receptors in the CeA were up-regulated at the 24- and 48-h stages, whereas SP receptors were down-regulated in the LC at the same stages. In concert with the literature indicating SP antagonist's antidepressive effects, up-regulated SP receptors in the CeA might contribute to the development of stress-related depression.