The ontogeny of muscarinic cholinergic binding sites was studied in rat fetal central nervous system by in vitro autoradiographic techniques using [3H]N-methyl scopolamine as ligand (1 nM). Nonspecific binding was determined after the addition of 1 microM atropine. The main findings of this study are the early appearance of muscarinic cholinergic binding sites in fetal rat central nervous system before gestational day 14, their subsequent spread in a caudofrontal direction and the rapid change of patterns within individual brain regions. Muscarinic cholinergic sites are present shortly after cell birth, though the time-lag between cell generation and expression of muscarinic sites differs between neuronal cell populations. High receptor densities are noted in certain brainstem nuclei that are important for early fetal and neonatal behaviors.