Tyrosine hydroxylase (TH) catalyzes the conversion of L-tyrosine to 3,4-dihydroxy-L-phenylalanine, which is the first and rate-limiting step in catecholamine biosynthesis. We have previously shown that the cyclic AMP response element (CRE), an essential promoter element for both basal and cyclic AMP-inducible TH transcription, activates the promoter activity in a distance-dependent manner. To identify further cis-regulatory elements controlling TH gene expression, we analyzed the potential regulatory sequences by several approaches. First, using transient transfection assays, we examined the cell-specific promoter activities of TH-reporter gene constructs and a dopamine β-hydroxylase (DBH)-reporter construct containing the 5′ upstream sequences of the rat TH and human DBH genes, respectively, that had been shown to direct tissue-specific reporter expression in transgenic mice experiments. Second, DNase I footprinting analysis of the 503-bp proximal area of the rat TH gene identified seven footprinted regions that encompass the putative cis-regulatory motifs, including the CRE (domain I), Sp1(domain III), Octamer (domain IV), AP1 (domain V), AP2 (domain VI), and two potentially novel sequence motifs (domains II and VII). Footprinting patterns at these sites by nuclear proteins from TH-positive and -negative cell lines appeared to be similar. Third, site-directed mutagenesis demonstrates that domain III, but not domain II, critically contributes to the TH promoter activity. Furthermore, electrophoretic mobility shift, competition, and supershift assays demonstrate that domain III is an authentic Sp1 site and that the transcription factor Sp1 interacts with it. This and previous results suggest that the CRE and Sp1 site may synergistically activate TH transcription in a promoter context-dependent manner.