The cholinergic innervation of the rat cerebral cortex was examined in pre- and postnatal life using immunohistochemistry with a monoclonal antibody directed against choline acetyltransferase (ChAT). Our observations show that there are two separate phases in the development of the cholinergic innervation of the rat neocortex. The first, a transient phase, occurs in the late stages of gestation and in the perinatal period. During this time, ChAT-labelled cells (neuroblasts, as well as immature pyramidal and non-pyramidal neurons) are present throughout the entire rostro-caudal extent of the primordial cortex. The fate of these cells, which are not visible shortly after birth, is unknown as is their functional role in the developing cortex. The second phase in the development of the cholinergic innervation begins in the middle of the second postnatal week. At this stage only a few faintly stained neurons and fibres appear in the cortex. Their numbers and staining intensity increase gradually until the fifth postnatal week when ChAT-labelled neurons and axonal arbours appear indistinguishable from their adult counterparts. The pattern of development observed in the second phase parallels closely that shown in a recent analysis of cortical ChAT activity during postnatal life.