The methylating enzyme catechol-O-methyltransferase (COMT) is an important inactivator of substrates containing catechol-structure, such as catechol neurotransmitters and hormones. In previous studies, the rat COMT gene has been cloned and characterized, and it has been shown that the two COMT polypeptides, S- and MB-COMT, are expressed from one gene by cooperation of two separate promoters. One promoter, P2, functions constitutively, whereas the other, the proximal P1 promoter, is regulated in a tissue-specific manner. In this report, a more detailed analysis of the rat P1 promoter is presented. By using reporter gene constructs, it is shown that upstream sequences of the P1 promoter contain several regions that modulate the expression either positively or negatively. These experiments also show that the region between the MB- and S-ATG translation initiation codons is indispensable for the activity of this promoter. Analysis of this region by DNase I footprinting and gel retardation assays identified the presence of several DNA elements with SP1 and NF1 recognition site homologies that bound both liver and brain nuclear proteins. However, one 11-nucleotide-long DNA region containing an overlapping consensus binding sequence for CREB and C/EBP-like factors reacted only with the liver nuclear lysate. Supershift experiments suggest that the transcription factor C/EBPalpha mediates the tissue-specific expression of the rat COMT P1 promoter.