The cerebral cortex of the mammalian brain has expanded rapidly during the course of evolution and acquired structurally distinguishable areas devoted to separate functions. In some brain regions, topographic restrictions to cell intermixing occur during embryonic development. As a means of examining experimentally whether such restrictions occur during formation of functional subdivisions in the rat neocortex, clonally related neocortical cells were marked by retroviral-mediated transfer of a histochemical marker gene. Clonal boundaries were determined by infection of the developing brain with a library of genetically distinct viruses and amplification of single viral genomes by the polymerase chain reaction. Many clonally related neurons in the cerebral cortex became widely dispersed across functional areas of the cortex. Specification of cortical areas therefore occurs after neurogenesis.