We have used the property of natural cell buoyant density to selectively fractionate embryonic rat neocortical cells into 20 subpopulations ranging in phenotype from proliferatively active progenitors to terminally postmitotic neurons. Immunocytochemical and cell cycle analysis of the cellular fractions with flow cytometry revealed an inverse relationship between cell buoyant density and neuronal differentiation. The most buoyant fractions contained predominantly terminally postmitotic, tubulin betaIII-positive, tetanus toxin-positive, and nestin-negative differentiating neurons, while immature, bromodeoxyuridine-positive and nestin-positive proliferating cells were more prevalent in less buoyant fractions. Double loading of isolated cells with voltage- and Ca2+-sensitive fluorescent indicator dyes followed by simultaneous recordings of membrane potential and cytoplasmic [Ca2+] ([Ca2+]c]) using flow cytometry revealed that >50% of the least buoyant cells produced functional responses to veratridine, a Na+ channel agonist, and muscimol, a GABA(A) receptor agonist, but <10% responded to kainic acid, an agonist of a subset of glutamate receptors. As cells became more buoyant the percentage of cells that depolarized and produced a rise in [Ca2+]c to each ligand increased, particularly in response to kainic acid. Short-term culture of select fractions revealed a marked enrichment for cells with morphologies and epitopes characteristic of neuronal and progenitor cell subpopulations. The results show that embryonic cortical cells exhibit a range of naturally occurring buoyant densities that can be used to expeditiously fractionate cortical cells according to their pre- or postmitotic status, thus providing ready access for cellular and molecular studies of proliferation and differentiation.