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 (ORE), an essential promoter element for both basal and cyclic AMP-inducible TH transcription, activates the promoter activity in a distancedependent 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 /3-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 ORE (domain I), Spi (domain Ill), Octamer (domain IV), APi (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 Ill, but not domain II, critically contributes to the TH promoter activity. Furthermore, electrophoretic mobility shift, competition, and supershift assays demonstrate that domain Ill is an authentic Spi site and that the transcription factor Spi interacts with it. This and previous results suggest that the ORE and Spi site may synergistically activate TH transcription in a promoter context-dependent manner. Key Words: Tyrosine hydroxylase-Dopamine /3hydroxylase-Transcriptional regulationc/s-Regulatory elements-Oyclic AMP response element-Transcription factor Spi -Oell-type specific transcription.