Pineal-specific expression of the tryptophan hydroxylase (TPH) gene has been demonstrated by a number of studies. However, little is known about the regulatory mechanism for pineal-specific expression of the TPH gene. To identify the cis-acting region responsible for pineal-specific expression of the TPH gene, we investigated a 6.1-kb 5'-flanking region of the mouse TPH gene using an immortalized pineal cell line (PGT-beta) derived from transgenic mice. By deletion analysis, it was demonstrated that the pineal-specific enhancing region resides approximately between -6.1 and -4.7 kb upstream from the transcription initiation site of the mouse TPH gene. Additionally, nucleotide sequence analysis of this region showed that the (AC/TG)22 repetitive sequence is located approximately -5.78 kb upstream of the mouse TPH gene, and several known tissue-specific cis-acting elements, such as Pit-1 and the pituitary specific element (PSE), have also been identified in the region. We believe that the analysis of the sequence and several cis-acting elements in the pineal-specific enhancing region of the mouse TPH promoter would enhance our understanding of the precise mechanism of pineal-specific expression.