The subclone HCN-1 was derived from parental cell lines from cortical tissue of a patient with unilateral megalencephaly growth and immunochemistry staining characteristics [G. V. Ronnett et al. (1990) Human cortical neuronal cell line: establishment from a patient with unilateral megalencephaly. Science 248, 603-605]. As we and others have shown, HCN-1A cells can be induced to differentiate to a neuronal-like morphology. HCN-1A cells stain positively for neurofilament, neuron-specific enolase and the low-affinity neurotrophin receptors, p75NGFR, but not for myelin basic protein, S-100, or glial fibrillary acidic protein (GFAP). HCN-1A cells also stain positively for gamma-aminobutyric acid and glutamate. In the present study, we examine the effects of cell density on the requirements for efficient induction of differentiation of HCN-1A cells and analyze the time course of this induction and its reversion. We also characterize the changes in cytoskeletal proteins of HCN-1A cells in response to their differentiation neuronal phenotype.