In the ventral mesencephalon of the E14 rat fetus, 90% of the dopaminergic, tyrosine hydroxylase positive (TH+) cells are localized in 1.0 mm3 of tissue. This same ventral mesencephalic region also contains 90% of the dopamine content of the E14 ventral brainstem (2.2 +/- 0.3 nmol/mg protein). When cells were prepared for culturing from this localized area, and plated at a density of 2.5 x 10(5) cells/cm2, 17-21% of the cells were TH+, at 4 and 12 h, and at 1, 5, 7 and 10 days after plating. The percentage of TH+ cells was also 17-21% when examined at 4 h, 12 h or 5 days after plating at densities ranging from 7.8 x 10(3) to 2.5 x 10(5) cells/cm2. However, cell survival at a density of less than 6.2 x 10(4) cells/cm2 was poor after 5 days in culture. Based on the degree of neurite elongation and complexity, cell maturation appeared to be complete at 5 days in culture (DIV5), and appeared to be maintained at this level up to DIV10. By DIV14, neurite retraction was evident, and the cells were more rounded. These signs may indicate the inception of senescence in the cultures. A benztropine-sensitive, concentration-dependent dopamine uptake mechanism was demonstrated in the cultures at DIV7, and DA could be released from preloaded cells using 50 mM K+. Five morphological subtypes of TH+ cells were identified in the cultures. This primary culture of the ventral mesencephalic, dopaminergic area, with a high percentage of TH+ cells, is suitable for use in acute biochemical and cellular studies, between DIV 5 and DIV10.