Detection and pharmacological characterization of OT-binding sites were performed on 12-day-old rat spinal cord membranes and on embryonic cultured spinal neurones and astrocytes after 12 days in culture. In neurone-enriched cultures, OT-binding sites were detected by autoradiography on cells morphologically comparable to neurone-specific enolase immunoreactive cells. In astrocyte cultures, as shown by combination of autoradiography and immunocytochemistry, OT-binding sites were detected on cells expressing the glial fibrillary acidic protein (a specific astrocytic marker). The pharmacological characterization was assessed by binding studies performed with a highly specific radioiodinated OT antagonist on postnatal rat spinal cord membranes and on embryonic cultured spinal cord neurones and astrocytes. The saturation studies suggested the presence of a single class of binding sites of high affinity for the OT antagonist on spinal membranes and cultured cells, as already described in the rat for central and peripheric OT receptors. The competition studies indicated that on spinal membranes, OT and AVP had the same high affinity, as classically described, whereas on cultured cells, AVP had a lower affinity, suggesting that culture conditions may influence the pharmacology of the spinal OT-binding sites. Involvement of NEM- and Gpp[NH]p-insensitive G-proteins in the coupling of the spinal OT-binding sites with the effector system was evidenced on 12-day-old rat spinal membrane preparations and on neurone and astrocyte cultures.