Parkinson's disease (PD) is characterized by the selective loss of midbrain dopamine neurons. Neural transplantation with fetal dopamine neurons can be an effective therapy for patients with PD, but recovery of human fetal cells is difficult. Scarcity of tissue has limited clinical application to a small number of research subjects worldwide. Selective differentiation of embryonic stem cells (ESCs) to dopamine neurons could lead to an unlimited supply of cells for expanded clinical transplantation. To facilitate the differentiation and purification of dopamine neurons, the green fluorescent protein (GFP) gene was inserted into the dopamine transporter (DAT) locus in mouse ESCs using homologous recombination. From these DAT-GFP ESCs, dopamine neurons expressing GFP were successfully produced by in vitro differentiation. The DAT-GFP ESCs were used to generate DAT-GFP knock-in mice. We have found that GFP was colocalized with DAT, Pitx3, Engrailed-1, and tyrosine hydroxylase-positive cells in midbrain, hypothalamus, and olfactory bulb but not in noradrenergic cell regions or other ectopic sites. The GFP-positive dopamine neurons could be isolated from embryonic day-15 ventral midbrain by fluorescence activated cell sorting. These purified dopamine neurons survived reculture and expressed tyrosine hydroxylase and DAT when cocultured with mouse astrocytes or striatal cells. Animals homozygous for DAT-GFP were hyperactive because they had no functional DAT protein. These DAT-GFP knock-in ESCs and mice provide unique tools for purifying dopamine neurons to study their physiology, pharmacology, and genetic profiles.