We recently established primary cultures from dissociated second trimester human fetal brains using a novel spin seeding method and characterized cellular populations with distinct phenotypes in these cultures. Here, we report that these neural cultures can be dissociated to single-cell suspensions, sorted by size using flow cytometry and re-seeded to yield cultures selectively enriched for the neuronal and glial cell populations. Sorted neurons were highly homogeneous, viable and extended processes, by one day after re-seeding. These neurons expressed immunoreactivity for neurofilament protein, retained their GABAergic phenotype and were electrically excitable. Re-seeded astrocytes proliferated in culture and expressed glial fibrillary acidic protein. We describe the conditions required for the flow cytometric sorting and tissue culture assays as well as the morphological, immunocytochemical and electrophysiological characteristics of the sorted neuronal population.