The specific binding of 125-Bolton Hunter substance P (125I-BHSP) was estimated on 4- to 5-week-old primary cultures of astrocytes from several brain structures and the spinal cord of 16-day-old embryonic or newborn rats. In both cases, high levels of binding of 125I-BHSP were found on intact astrocytes from the brainstem, but this binding was low or negligible on cells from the cerebral cortex, striatum, hypothalamus, and mesencephalon. In addition, hippocampal astrocytes from newborn rats were also devoid of 125I-BHSP binding sites, while a binding of 125I-BHSP (half that of brainstem cells) was observed on astrocytes from the cerebellum and spinal cord. It was also shown that this regional heterogeneity in 125I-BHSP binding was not linked to differences in the inactivation of the ligand, cell plating density. or eventual cell contaminants. Five-day-old cultures from 16-day-old embryos were used to estimate 125I-BHSP binding on neuron-enriched cultures. Specific 125I-BHSP binding was found on cells from the brainstem, mesencephalon, and hypothalamus, but neurons from the cerebral cortex or the striatum contained low or negligible amounts of 125I-BHSP binding sites. Competition studies using tachykinins and SP analogues indicated that 125I-BHSP binding sites on brainstem astrocytes (16-day-old embryos) have the pharmacological profile expected for NK1 binding sites. SP (1 μM) stimulated phosphoinositide breakdown in cells rich in 125I-BHSP binding sites (brainstem) but not in those devoid of 125I-BHSP binding (striatum). These results are discussed in light of previous observations that have shown the presence of 125I-BHSP binding sites on astrocytes from different brain structures of the mouse.