Cultures of PC 12 pheochromocytoma cells were established on a polyornithine substratum in medium supplemented with the chemically defined NI mixture in the presence or absence of Nerve Growth Factor (NGF). Normal cell proliferation in the absence of NGF was cqually competent when fctd calf scrum (FCS) was replaced with N I-supplemcnted medium. The differentiation of PC 12 cells. which occurs upon NGF treatment, ultimately results in cell death without the addition of 0.1 % FCS to the Nl-supplemented medium. The combination of N I , 0.1% FCS, and NGF permits the PC12 cells to develop a neuritic outgrowth much earlier than when higher (I-lo%,) FCS levels are used. Neurite retraction is caused in a dose-dependent manner by a delayed presentation of FCS. Within 2 days of serum presentation, however, neurites regrow to achieve that percentage of neurite-bearing cells which is seen without a serum challenge. Moreover. the retraction response becomes less pronounced with time over the &day culture period for any given serum concentration. Among the N1 ingredients, only insulin and transferrin are needed by PC 12 cells for survival whether in the dividing state or not. Neurite growth was not dependent on any of the N1 components.