Expression of tyrosine hydroxylase (TH) is limited to catecholamine-producing neurons and neuroendocrine cells in a cell type-specific manner and is inducible by the cAMP-regulated signaling pathway. Previous results indicated that the cAMP response element (CRE) residing at -45 to -38 base pairs upstream of the transcription initiation site is essential for both basal and cAMP-inducible promoter activity of the 2.4-kilobase or shorter upstream sequence of the TH gene (Kim, K. S., Lee, M. K., Carroll, J. , and Joh, T. H. (1993) J. Biol. Chem. 268, 15689-15695; Lazaroff, M. , Patankar, S., Yoon, S. O., and Chikaraishi, D. M. (1995) J. Biol. Chem. 270, 21579-21589). Here, we further report that the CRE is critical for the promoter activity of the 5.6- or 9.0-kilobase upstream sequences of the rat TH gene, which had been shown to direct the cell-specific TH expression in vivo. To define the structure/function relationship of the CRE in transcriptional activation of the TH gene, we performed saturated mutational analyses of 12 nucleotides encompassing the CRE. Mutation of any nucleotide within the octamer motif results in a significant decrease of both basal and cAMP-inducible transcriptional activity of the TH reporter gene construct. Among the four nucleotides adjacent to the CRE (two 5' and two 3'), only the G residue at the immediate 3' position is important for full transcriptional activity. DNase I footprint analysis indicates a positive correlation between in vivo promoter activity and in vitro interaction between the CRE motif and its cognate protein factor(s). Reconstruction experiments using a TH promoter in which the native CRE was rendered inactive show that the CRE can transactivate transcription in either orientation through a window of approximately 200 base pairs upstream of the transcription initiation site, suggesting that CRE supports transcriptional activation of the TH gene in a distance-dependent manner. Finally, when the distance between the CRE and TATA box was changed by inserting an additional 5 or 10 bases, it was observed that both insertional mutations increased activity by approximately 3-fold. The cAMP inducibility was as intact as the wild type construct. Together, these results are consistent with a model in which transcriptional activation of the TH gene by the CRE requires that it be located within a certain proximity of the CAP site but does not depend on a stringent stereospecific alignment in relationship to the TATA element.