The ontogeny of binding sites for [3H] spiperone was studied in time-pregnant rats. Binding of [3H]spiperone to fresh homogenates of pre- and postnatal rat forebrain was characterized by Scatchard analysis and competition experiments with a number of dopaminergic and serotonergic agonists and antagonists and additional substances. A convenient discrimination of three high-affinity sites, i.e., the dopaminergic D2, serotonergic S2, and spirodecanone (Sd) sites, was obtained with l-(-)sulpiride and cis-flupenthixol. The analgesic R5573 was found not to be specific for the Sd site but to interact with all three sites. The three binding sites became detectable in sequential order. S2 and D2 binding sites were first found at embryonic days 15.75 and 17.75, respectively. The Sd site did not appear before postnatal day 8. All three binding sites reached adult values at approximately postnatal day 30. During the prenatal period, the increase in the number of D2 binding sites paralleled the rise in forebrain dopamine concentrations. The kinetics of D2 and S2 sites were the same at embryonic day 19.75 and postnatal day 30. These observations provide evidence for the presence of the receptor substrate for actions of neuroleptics on dopaminergic and serotonergic systems during fetal life.