Actively dividing cells cultured from human leptomeninges obtained at autopsy and from human pia obtained at neurosurgery expressed two antigens characteristic of a mature neuronal phenotype: the 160 kDa neurofilament protein (NF-M) and neuron-specific enolase (NSE). The cells lacked typical glial, epithelial and endothelial markers but did contain vimentin, a protein normally associated with but not limited to mesenchymally derived cell types. Immunocytochemical results using redundant antibodies were consistent over serial passage and were confirmed by Western blot analysis. Morphologically the cells were pleiomorphic with frequent long, NF-M + processes. The antigenic characteristics of the cultured leptomeningeal and pial cells were similar to those exhibited by human autopsy and biopsy brain-derived cultures described previously, suggesting that the latter may have originated from pial cells adherent to the autopsy and biopsy brain tissue specimens.