Tyrosine hydroxylase (TH) is selectively expressed in catecholaminergic neurons and in chromaffin cells of the adrenal medulla. Constructs in which 5' flanking sequences of the rat TH gene directed expression of bacterial chloramphenicol acetyltransferase (CAT) were transfected into cell lines and assayed for transient expression of CAT. In most nonexpressing cell lines, CAT levels were less than 5% of that found in a TH-positive pheochromocytoma line (PC8b). In two lines described here, a rat anterior pituitary cell line (GH4) and a rat fibroblast line (Fr3T3), CAT expression reached 12 and 20%, respectively, of the PC8b level. Greater than 90% of the PC8b activity was lost when sequences between -212 and -187 (in relation to the transcriptional initiation site) were deleted. Further deletions that removed the cyclic AMP response element (CRE) (-45) and the TATA box at -29 reduced transcriptional activity to background in all three lines. These data suggest that 212 nucleotides of the 5' sequence are sufficient for pheochromocytoma expression and that information between -212 and -187, which includes an AP1 site (-206 to -200), is essential for full transcriptional activity. In addition, sites for other protein transcription factors (AP2, POU/Oct, SP1, and CRE) reside between -221 and -38 and are largely conserved between the human and rat gene.