Neurons of the mammalian cerebral cortex are commonly subdivided into two broad classes: pyramidal and nonpyramidal. The former are projection neurons, while the latter are interneurons. To determine whether the two neuronal classes in the cerebral cortex are derived from the same or separate progenitor cells, we used a recombinant retrovirus containing the reporter gene E-coli beta-galactosidase as a lineage marker. Clonally related neurons expressing the inherited beta-galactosidase gene were detected histochemically, at both light and electron microscopic levels, and their phenotypes were identified using well-established ultrastructural criteria. The clones examined, with one exception, were composed of either all pyramidal or all nonpyramidal neurons. These findings suggest that pyramidal and nonpyramidal neurons in the cerebral cortex have separate lineages and are derived from different progenitor cells in the ventricular zone. This lends weight to the notion that cells in the ventricular zone comprise a heterogeneous population, and that lineage contributes substantially to the phenotype of a neuron.