We have screened lines of nonpassaged epidermal growth factor-responsive neurospheres from embryonic striatum and brainstem. They have been maintained in defined medium with epidermal growth factor over a period of 2 years and remained in an undifferentiated state to this date. Since isolation from the brain 2 years ago, these nonpassaged epidermal growth factor responsive neurospheres have shown active proliferation and self-renewal capacity. When subplated on a poly-D-lysine coated surface, they resumed differentiation within 24 hours. The differentiation process of the nonpassaged epidermal growth factor responsive neurosphere appeared to recapitulate the neural development in the brain. Many cells migrated, extending radial processes while expressing nestin and S100 in the early 7-day subplating culture. They continued to differentiate into major neural types in 14-day subplating culture, including fibrous and cytoplasmic astrocytes, oligodendrocytes, and serotonin, gamma-aminobutyric acid, and a small number of tyrosine hydroxylase-positive neurons. The nonpassaged epidermal growth factor-responsive neurospheres in many ways resemble hemopoietic cells. Both are proliferative, possess the potential of indefinite self-renewal, yet multipotent, and are capable of resuming the differential pathway. The nonpassaged epidermal growth factor responsive neurospheres meet the criteria of stem cells and have been found to be a useful model to study the development in vitro.