Development of the dopamine (DA) neuron phenotype was monitored in cultures of embryonic rat mesencephalon (MES) and hypothalamus (HYP) maintained for 1 to 21 days in vitro (DIV) in the absence of glial support cells. Cell counts following immunohistochemistry for tyrosine hydroxylase (TH) demonstrated that the number of DA neurons declined by 85% in MES cultures yet increased 5-fold in cultures of HYP, so that by 21 DIV equal numbers of DA neurons were present in these culture systems. After 21 DIV MES DA neurons exhibited a multipolar morphology, with numerous branching processes. HYP DA neurons were primarily fusiform in shape with fewer processes and process branch points. Double-label immunohistochemistry for TH and microtubule-associated protein 2 identified the majority of TH-positive processes in either culture system as dendrites. Individual MES but not HYP DA neurons were also found to generate axons. Western analysis showed that between 1 and 21 DIV the concentration of TH protein increased 2-fold in MES and 4-fold in HYP cultures. After 21 DIV the concentration of TH protein in MES cultures was twice that found in cultures of HYP. In the period between 1 and 21 DIV levels of tetrahydrobiopterin (BH4) increased by 6-fold in MES and 20-fold in HYP cultures. After 21 DIV BH4 content was 3-fold higher in HYP than in MES cultures. The abundance of the mRNA encoding for GTP cyclohydrolase I, the rate-limiting enzyme in BH4 biosynthesis, was similar in MES and HYP cultures despite this difference in BH4 levels. In contrast, TH mRNA was 4-fold more abundant in MES than in HYP cultures. Treatment of MES cultures with the DA neuron toxin 1-methyl-4-phenylpyridinium decreased DA cell numbers, TH protein content and BH4 levels, demonstrating that BH4 is localized primarily to DA neurons. Similar treatment of HYP cultures did not effect any of these parameters. Steady-state levels of DA and the rate of DA synthesis were both 3-fold higher in MES than in HYP cultures. A 95% decline in BH4 content produced by inhibiting BH4 biosynthesis resulted in 64% and 84% declines in the rate of MES and HYP DA synthesis, respectively. Overall, these observations indicate that, with the exception of the capacity to synthesize DA, DA neurons in MES and HYP cultures share few common properties.