Expression of tyrosine hydroxylase (TH) is regulated in a tissue-specific manner by multiple mechanisms. In catecholaminergic cells, the expression of TH-mRNA is up-regulated by forskolin (FK) and is suppressed by retinoic acid (RA). We have previously provided evidence that, in N-18 cells, the expression of TH-mRNA is suppressed by DNA methylation of the TH gene itself. In the present study, using a catecholaminergic cell line, N1E-115, we performed deletional and mutational analyses on the 5'-flanking region of the mouse TH gene. The results indicate that a cAMP response element (CRE) mediates constitutive transcription of the TH gene, as well as responsiveness to FK and RA. Using bisulfite sequencing methods, we analyzed the methylation status of the TH gene 5'-flanking region in various cell lines and rat tissues. We found that three cytosine residues in the domain surrounding the CRE of the TH gene promoter were specifically methylated in N-18 cells and TH non-expressing rat tissues. In contrast, these cytosines were undermethylated in TH expressing cell lines and tissues. The inverse correlation between the frequency of cytosine methylation at these specific sites and the levels of TH expression supports a role for DNA methylation in the regulation of tissue-specific gene expression.