Primary midbrain cultures are valuable for probing the function of dopaminergic neurons and for elucidating the factors that cause their dysfunction and degeneration. To allow more effective control of the culture environment, we have characterized the survival, differentiation, and trophic factor response of dopaminergic neurons in the absence of serum. Combinations of media and supplements markedly affected all three indices measured. Combinations that produced maximal dopaminergic neuron survival are different from those that result in maximal differentiation and trophic factor response. Furthermore, antioxidant treatment was effective with only one medium/supplement combination, indicating that these neurons were not degenerating as a result of oxidative stress in the majority of culture conditions used in this study. These results demonstrate that dopaminergic neurons can be grown in serum-free conditions but that the choice of culture conditions has a marked influence on cell survival and function.