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Conditions for long-term cultivation of human fetal brain cells in a chemically defined medium were established using cryopreserved brain fragments obtained from legal abortions. Tissue of the same gestational age was pooled and the cells cultured in a fully defined medium containing insulin-like growth factors (IGF I and II). Primary cultures were kept for 2-4 weeks and secondary or tertiary cultures could be maintained for 3 months. The cultures were characterized by morphological, electrophysiological and biochemical methods. Glial cells were predominant during the first two weeks of culture. In later stages of cultivation, glial cells diminished in number and most cells were neuronal. Voltage-dependent Na+ channels were recorded from neurons. Biochemical studies indicated that the fetal brain cells contained and secreted immunoreactive somatostatin as well as the tachykinins, substance P and neurokinin A. Cultures grown in IGF II- or nerve growth factor-containing medium expressed increased choline acetyltransferase activity.