Development of central nervous system pathways involves the action of soluble factors on target cells and interactions between various cell types. One of the best defined CNS pathways is the nigrostriatal pathway. To examine events involved in the formation of this pathway, development of dopaminergic neurons from the embryonic rat mesencephalon was examined in dissociated cell cultures. Cells were grown in serum-free medium or serum-containing medium in the presence or absence of the basement membrane component, laminin. Mesencephalic cells were obtained from embryonic day 13 to embryonic day 15 rat brains; cells from other regions were obtained from embryonic or postnatal rat brains at times when subsequent in vitro development was shown to be optimal. Development of the dopaminergic neurons in the mesencephalic cultures was determined by uptake of exogenous, labelled dopamine. Mesencephalic dopaminergic neurons grown alone developed vigorously in culture for 9 days. After this time development ceased. In contrast, if mesencephalic neurons where grown in the presence of cells from other brain regions, the cultures continued to develop to day 20 or day 27. Glial cells from these other brain regions appeared to be important for the continued development of the mesencephalic neurons. There also appeared to be a later stage of development or cell survival that was dependent on the presence of specific target cells in the culture. Striatal neurons are the normal in vivo target for most of the mesencephalic dopaminergic neurons. Cells from the striatum provided optimum in vitro conditions for development of the dopaminergic neurons.