The enzyme catechol-O-methyltransferase (COMT) catalyzes the inactivation of catechol-containing molecules by methylation. The cDNAs for the rat and human COMT have recently been cloned and recombinant proteins expressed in prokaryotic and eukaryotic cells. We describe here the structure of the rat COMT gene and its 5'-flanking sequences. The gene spans at least 13 kb and is composed of 5 exons, the first one noncoding. The two ATG codons for the initiation of translation of the membrane-bound (MB-COMT) and soluble (S-COMT) forms of the enzyme reside in the second exon. The gene expresses two mRNA species of 1.6 kb and 1.9 kb that have different tissue distributions. The expression of the transcripts is regulated by at least two promoters, P1 and P2. The P1 promoter expresses the shorter transcript in a tissue-specific manner and is located between the ATG codons in the coding region of the longer transcript. The P2 promoter is constitutive and responsible for the expression of the longer transcript. The shorter 1.6-kb mRNA (S-mRNA) produces only the S-COMT polypeptide, whereas the longer 1.9-kb mRNA (MB-mRNA) is able to direct synthesis of both forms of the COMT enzyme.