We have developed a pure cortical neuronal culture free of glial cells, grown in a serum-free environment. The cultured cells immunostained positively with neurofilament antibody while they displayed virtually no glial cell characteristics, such as glial fibrillary acidic protein, glycerol phosphate dehydrogenase or glutamine synthetase. Insulin and transferrin were necessary and sufficient for neuronal survival, neurite extension and glutamic acid decarboxylase (GAD) expression. Insulin-like growth factor-I was able to replace insulin and was active close to its physiological concentration, suggesting it might be the in vivo factor influencing neuronal growth in the brain. The dynamics of the developmental process were striking. The neurons moved on the poly-D-lysine covered plastic dish, and rearrangements in contacts between cells were observed. At first the neurons underwent a general cellular growth manifested by a large increase in the culture total protein content and by the initiation of neurites. A more specific differentiation, as indicated by the sharp increase in GAD levels which was concurrent with an increase in interneuronal contacts, lagged behind the initial growth. Thyroid hormone (TH) affected the differentiation process, causing a future increase in GAD levels during the same time of increase in neurite growth, in interneuronal contacts, in thyroid hormone receptors and thyroid gland maturation. Removal of each of the hormones after a few days of cell growth revealed that transferrin was still required for neuronal survival while insulin became essential for general cellular growth but not specific neuronal differentiation, since it caused an increase in both the total protein and GAD levels but not in GAD specific activity. TH, on the other hand, affected the differentiation process as evident by its ability to increase GAD specific activity. This action of TH, however, required the presence of insulin, without which no increase in GAD level by TH was observed. This neuronal culture, glial and serum-free, provides a new system for investigating neuronal development and function in the complex mammalian central nervous system.