The fine structure, synaptic relationships, distribution and time of origin of interstitial neurons situated within the white matter subjacent to the visual, somatosensory and motor cortices were studied in the human and monkey telencephalon. The analysis was carried out on Nissl-stained serial sections, rapid Golgi impregnations, by acetylcholinesterase (AChE) histochemistry, electron microscopy and [3H]thymidine ([3H]TdR) autoradiography. Interstitial neurons have a similar distribution, morphology and histochemistry in both human and monkey telencephalon. Their highest density and the most extensive distribution is found in the neonatal period in both species. The number of interstitial neurons decreases during infancy, but numerous cells remain in the adult. Two types of interstitial neuron can be recognized in Golgi preparations: polymorphic cells, usually situated close to the cortex and fusiform cells, located predominantly in the depths of the white matter. The polymorphic cell type is prevalent during neonatal and infant stages, while fusiform cells are relatively more numerous in the adult. Interstitial cells have ultrastructural features and organelles typical of neurons of the central nervous system with well-defined axosomatic and axodendritic synapses of both symmetrical and asymmetrical types. About 20% fo the interstitial cells show strong specific AChE activity. Autoradiographic analysis of postnatal monkeys exposed to [3H]TdR at various embryonic (E) and early postnatal days indicates that interstitial neurons which lie beneath the visual and somatosensory-motor cortices are generated between E38 and E48. Contrary to the prevailing notion that interstitial neurons are the latest generated cells arreste during migration across the maturing white matter, they prove to be produced at the end of the first third of the 165-day gestation in the rhesus monkey concomitantly with the generation of neurons destined for the deep neocortical layers. These findings raise the possibility that interstitial cells represent a vestige of the transient embryonic subplate layer.