By combining [3H]thymidine autoradiography with choline acetyltransferase (ChAT) immunocytochemistry, we have determined the generation pattern of the large cholinergic neurons in the neostriatum. All of these neurons are produced between embryonic days 12 and 17 (E12-E17), with 75% of them being born between E13 and 15. Cholinergic neurons appeared to be among the earliest cells produced in the neostriatum when compared with previous generation studies of all neurons in the rat caudate-putamen. The caudal-to-rostral neurogenic gradient reported in previous investigations of all neurons was the only spatiotemporal gradient observed for cholinergic neurons. The generation peak for these cells was E13 caudally, and E15 rostrally. Additional immunocytochemical studies detected ChAT immunoreactivity within somata and primary dendrites of 1 day postnatal (1 dpn) rat neostriatum, and subsequently demonstrated a dramatic increase in the intensity of reaction product and the complexity of dendritic arborizations by 14 dpn. Large ChAT-positive neurons of the basal forebrain contained within the same specimens appeared to differentiate their cholinergic phenotype earlier than those in the neostriatum. However, recent generation studies of basal forebrain neurons combined with the present results have demonstrated that both cholinergic populations are produced simultaneously along the same neurogenic gradients. This then represents an example of cholinergic projection (basal forebrain system) and local circuit (neostriatum) neurons that share similar generation patterns but differ with respect to sequences of transmitter phenotype expression. Thus, for cholinergic forebrain neurons, a cell's position along the neurogenic gradient and its transmitter phenotype appear to be more closely associated with its birth date than its ultimate projection or rate of differentiation.