The aim of this study was to examine the development of the basalocortical pathway by using choline acetyltransferase and nerve growth factor receptor immunocytochemistry, acetylcholinesterase histochemistry and retrograde axonal transport. The observations were made in the ferret because in this species brain development occurs over a much more protracted period than in the rat. Staining for choline acetyltransferase immunoreactivity in the brain was minimal before birth. Adult levels of staining for the enzyme were not seen in cell bodies until three weeks after birth and in axons up to six weeks after birth. This, however, did not mean that presumptive cholinergic pathways are absent early in development. There was strong staining for nerve growth factor receptor in basal forebrain neurons from at least two weeks before birth. Positive staining for acetylcholinesterase was found in axons that begin to invade the cerebral cortex a week before birth. The retrograde axonal transport technique showed that the basalocortical pathway has a normal organization in the neonate. The conclusion is that cholinergic pathways form early in the prenatal period in the ferret but express their transmitter function late in postnatal development.