The development of cholinergic terminals in rat brain has been quantitatively analyzed by [3H]hemicholinium-3 autoradiography. [3H]Hemicholinium-3 binds to high affinity choline transport sites, a specific marker for cholinergic neurons. In neonatal animals, kinetic and pharmacologic binding characteristics and regional distribution of [3H]hemicholinium-3 sites are consistent with specific cholinergic localization, as in the adult. The distribution of cholinergic terminals is described in the adult rat brain and during development, including heterogeneity of binding within several regions such as the striatum, nucleus accumbens, olfactory tubercle, cortex, and hippocampus. Early development and maturation vary greatly between brain regions. At embryonic day E18 and day 0, specific binding density is high only in the medial habenula. Development occurs primarily during the postnatal period in most brain regions examined. Many brain regions exhibit a lull in development between days 5 and 10, although the rate of development is highly region specific. Specific binding increases 2-12-fold between day 5 and adult animals, with adult density being achieved anywhere from day 15 to after day 21. The ontogeny of [3H]hemicholinium-3 binding sites generally occurs in a rostral to caudal direction. In the striatal body the characteristic lateral to medial gradient of binding site density is apparent by day 5, and development is more rapid in the lateral striatum. Patches of dense [3H]hemicholinium-3 binding coincident with acetylcholinesterase are observed on day 5 in the caudal striatum. The various patterns of cholinergic terminal development suggest that factors regulating cholinergic development are regional and complex.