We analyzed the molecular mechanisms involved in the acquisition and maturation of dopaminergic (DA) neurons generated in vitro from rat ventral mesencephalon (MES) cells in the presence of mitogens or specific signaling molecules. The addition of basic fibroblast growth factor (bFGF) to MES cells in serum-free medium stimulates the proliferation of neuroblasts but delays DA differentiation. Recombinant Sonic hedgehog (SHH) protein increases up to three fold the number of tyrosine hydroxylase (TH)-positive cells and their differentiation, an effect abolished by anti-SHH antibodies. The expanded cultures are rich in nestin-positive neurons, glial cells are rare, all TH+ neurons are DA, and all DA and GABAergic markers analyzed are expressed. Adding ascorbic acid to bFGF/SHH-treated cultures resulted in a further five- to seven-fold enhancement of viable DA neurons. This experimental system also provides a powerful tool to generate DA neurons from single embryos. Our strategy provides an enriched source of MES DA neurons that are useful for analyzing molecular mechanisms controlling their function and for experimental regenerative approaches in DA dysfunction.