Degeneration of the dopaminergic neurons of the substantia nigra is a hallmark of Parkinson's disease. To facilitate the study of the differentiation and maintenance of this population of dopaminergic neurons both in vivo and in vitro, we generated a knock-in reporter line in which the yellow fluorescent protein (YFP) replaced the first exon and the first intron of the tyrosine hydroxylase (TH) gene in one allele by homologous recombination. Expression of YFP under the direct control of the entire endogenous 5' upstream region of the TH gene was predicted to closely match expression of TH from the wild type allele, thus marking functional dopaminergic neurons. We found that YFP was expressed in dopaminergic neurons differentiated in vitro from the knock-in mouse embryonic stem cell line and in dopaminergic brain regions in knock-in mice. Surprisingly, however, YFP expression did not overlap completely with TH expression, and the degree of overlap varied in different TH-expressing brain regions. Thus, the reporter gene did not identify functional TH-expressing cells with complete accuracy. A DNaseI hypersensitivity assay revealed a cluster of hypersensitivity sites in the first intron of the TH gene, which was deleted by insertion of the reporter gene, suggesting that this region may contain cis-acting regulatory sequences. Our results suggest that the first intron of the rodent TH gene may be important for accurate expression of TH.