The diverse array of neurons and glia in the mammalian cerebral cortex arises from proliferating cells of the ventricular zone that surrounds the lateral ventricles of the developing brain. A fundamental but unresolved question is whether the individual cells of the ventricular zone are committed to producing progeny of only one particular phenotype or whether they generate progeny of more than one phenotype. We have begun to address this question by asking if individual cells of the ventricular zone generate exclusively neurons or glia at the onset of cortical neurogenesis in the rat. To assess the phenotypes of cells derived from a common progenitor cell, retroviral-mediated gene transfer was used to introduce the reporter gene, Escherichia coli beta- galactosidase, into ventricular zone cells at embryonic day 15 or 16. We used histochemistry to reveal beta-galactosidase-expressing cells in the mature rat cerebral cortex. Isolated clusters of beta-galactosidase- expressing cells, presumably clones, were identified in serial sections. Since the histochemical reaction product is electron dense, each cell could be examined at the ultrastructural level and assigned definitively to one of the major classes of cells in the cerebral cortex on the basis of well-established morphological criteria. This approach overcomes the problems of cell type identification encountered with light microscopy, where it is not always possible to distinguish between different cell phenotypes. We found that virtually all clones contained cells of exclusively one type: either all astrocytes, all oligodendrocytes, or all neurons. Furthermore, each particular cell type exhibited a different pattern and intensity of staining. The neuronal clones, with one exception, were composed of either all pyramidal cells (projection neurons), or all nonpyramidal cells (interneurons). The size and composition of neuronal clones did not seem related to their position in the cerebral cortex. Collectively, our observations indicate that separate progenitor cells exist for pyramidal neurons, nonpyramidal neurons, astrocytes, and oligodendrocytes. The striking phenotypic homogeneity in the clones arising from individual progenitor cells suggests that by the onset of cortical neurogenesis, at least some lineage restrictions have already occurred among the precursor cell population. Thus, our results suggest that lineage may play a pivotal role in determining some of the functionally important phenotypic attributes of cells in the cerebral cortex.