Biochemical analysis of the binding of [125I]Bolton-Hunter coupled substance P [( 125I]BH-SP) to slide-mounted sections of rat brain demonstrated that [125I]BH-SP labels a binding site with a structure-activity profile characteristic of a substance P receptor. Under optimized preincubation and incubation conditions, the locations of substance P (SP) receptors were visualized by film and emulsion autoradiography. Receptor densities were quantified by computer-assisted densitometry. SP receptors are widely but discretely distributed throughout sensory, limbic and cortical areas of rat brain, though several motor areas also possess SP receptors. No receptors were detected in the substantia nigra and interpeduncular nucleus, which are innervated by SPergic nerves; these regions of the brain may possess a low affinity SP receptor not detectable with this assay. Findings are discussed in the framework of an overall notion of the role of neuropeptides in the biochemistry of emotion.