The ontogeny of high affinity nicotinic cholinergic binding sites was studied in Long-Evans rat brain by in vitro autoradiography, using [3H]nicotine (10 nM) and cold (-)nicotine bitartrate to assess specificity. The first binding sites become detectable in spinal cord and caudal medulla oblongata at gestational day (GD) 12. Until GD 14, labelling spreads throughout lower brainstem, mesencephalon and parts of diencephalon, with higher densities in ventral areas (including the area of developing mesencephalic dopamine neurons). Matrix zones remain unlabelled. Receptor sites appear in the cerebellar anlage by GD 15, and in caudal caudate-putamen by GD 16. During development from late gestational to early postnatal stages, labelling is reduced in many lower brainstem areas and increases in forebrain, in particular in neocortex. Receptor density remains high in thalamus. In neocortex, nicotinic receptor sites are first seen in the subplate layer by GD 20. Labelling of this zone remains prominent until PN 14, when an additional band of increased receptor density is seen in cortical layers III/IV which contain high receptor levels in adulthood. At PN 27, the pattern has become similar to the adult one. The development of [3H]nicotine-binding sites in individual brain regions, with a general caudo-rostral gradient, accompanies cell differentiation and early synapse formation, e.g., in neocortex. The ontogenetic pattern differs in detail from that of muscarinic-cholinergic binding sites. The early presence of binding sites provides a basis for specific actions of nicotine on the fetal brain. As a consequence of the ontogenetic changes, different brain structures become targets for the action of this drug at different stages of development.