Tyrosine hydroxylase (TH), the first and rate-limiting enzyme in the biosynthesis of catecholamine neurotransmitters, is expressed within central and peripheral catecholaminergic cells. To delineate DNA sequences necessary for tissue-specific expression of the rat TH gene, transgenic mice were produced containing 0.15 kb, 2.4 kb, and 9.0 kb of 5' flanking sequence fused to the E. coli lacZ (beta-galactosidase) reporter gene. The reporter gene expression in the transgenic animals was monitored by both X-gal histochemical staining and beta-galactosidase immunohistochemistry and compared to TH mRNA and protein expression. Transgenic mice bearing 9.0 kb, but not the smaller constructs with either 2.4 kb or 0.15 kb of 5' flanking sequence, fused to lacZ were able to direct high level expression of beta-galactosidase at levels equivalent to the endogenous TH in central catecholaminergic cells, and to a lesser degree to adrenal gland. Previously, 4.8 kb of 5' flanking region was reported to contain some tissue-specific element(s) determined by chloramphenicol acetyltransferase (CAT) assay using regional brain dissections and was not able to demonstrate cellular localization of the CAT expression [2]. Using histological procedures which allow for spatial resolution, this study demonstrated that the crucial catecholaminergic neuron-specific DNA element(s) resides between -9 kb and -2.4 kb of the 5' flanking region of the rat TH gene; this assertion is substantiated by the high-level of tissue-specific expression of lacZ in catecholaminergic cells.