The development of a dopamine- and adenosine 3':5'-monophosphate-regulated phosphoprotein with an apparent Mr of 32,000 (DARPP-32) has been investigated in the central nervous system of the prenatal and newborn rat by immunocytochemical methods. DARPP-32 first appears in the rat brain at day 14 of gestation, in the anlage of the primary olfactory cortex and the caudate nucleus. Over the next few days, the number of immunoreactive cell bodies in these 2 areas, and in the olfactory tubercle and frontal cortex, increases rapidly. By the day of birth, most of the brain regions that will ultimately contain DARPP-32-positive somata already display a disposition toward DARPP-32-like immunoreactivity similar to that observed in the adult animal. In addition to the nuclei mentioned above, DARPP-32-containing cell bodies also appear over the intervening period in the olfactory nucleus, nucleus accumbens, central amygdaloid nucleus, lateral funiculus, and the choroid plexus and ependymal layers of the third, fourth, and lateral ventricles and the Sylvian aqueduct. Many of these immunoreactive cells disappear during subsequent postnatal maturation. DARPP-32-immunoreactive fibers were also observed in the prenatal and newborn rat CNS. As in the adult, the processes were observed in known target areas of the DARPP-32-containing neurons, namely, the globus pallidus, ventral pallidum, internal capsule, and substantia nigra. The ontogeny of tyrosine hydroxylase (TH)-like immunoreactivity was analyzed simultaneously. Of particular interest was the observation that the arrival within a given brain region of the presumed dopaminergic, TH-containing innervation, part of whose postsynaptic function is putatively mediated by DARPP-32, was preceded by at least 2 d by the appearance of the DARPP-32-containing cells. Moreover, the subsequent reorganization of the DARPP-32-positive somata within the caudate nucleus into distinct clumps also predated by 1 or 2 d the aggregation of the TH fibers into the same microzones. The development of DARPP-32-like immunoreactivity is mostly complete by the day of birth, and is consistent with its playing a role in mediating some of the postsynaptic actions of dopamine pathways. The appearance of this protein does not seem to be dependent on the presence of a dopaminergic innervation.