Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of catecholamines. We describe here the isolation of the chicken TH gene and the analysis of 3 kb of its 5' flanking region. The chicken TH transcription unit spans 19 kb. The 60-bp proximal promoter contains a TATA box and a cyclic AMP response element (CRE) sequence. The 5' flanking region contains several AP1-, AP2-, and octamer-like sequences as well as a glucocorticoid response element at position -1.4 kb. A construct containing the 3-kb 5' flanking DNA fused to the chloramphenicol acetyltransferase (CAT) gene was transiently transfected into PC12 cells, and the effect of various effectors was tested. Only forskolin increased the CAT activity, likely owing to the presence of the CRE sequence. Constructs prepared by progressively deleting the 5' flanking DNA were transfected into PC12 and QT6 (quail transformed fibroblasts) cells. In both cell types, the transcriptional activity increased with deletion of the 5' flanking region. These results show that the 60-bp region containing the TATA box and the CRE is sufficient to act as a constitutive promoter for the chicken TH gene and that this region appears to be negatively controlled by upstream sequences.