The expression of the catecholamine biosynthetic enzyme, tyrosine hydroxylase (TH), is confined to several different types of neuroendocrine cells. Using a transient assay system, we examined more than 10 kb of the human TH gene and 6.5 kb of 5' flanking sequences of the rat TH gene for DNA elements that confer cell-type specific expression. Surprisingly, these elements do not appear to be conserved in position or sequence across species. When plasmids containing DNA sequences - 749 bp from the transcription start site of the rat gene were introduced into PC12 cells, up to sixfold higher levels of expression were observed as compared to the same fragments introduced into HepG2 cells or LAN-1 cells. In contrast to the rat gene, analogous fragments of the human 5' promoter failed to confer cell-type specific expression. However, when plasmids containing a truncated thymidine kinase promoter and either orientation of a 760 by 3' human TH gene fragment were introduced into PC12 and LAN-1 cells, we observed a six- and 3.5-fold increase, respectively, over that observed for HepG2 cells. Subsequent deletion of this fragment led to significant activation of transcription in PC12 and HepG2 cell lines. These data indicate the presence of multiple elements contributing to the cell-type specific expression of tyrosine hydroxylase genes.