The coding sequence of rat liver catechol-O-methyl-transferase (COMT; EC 2.1.1.6) was determined from rat cDNA and genomic libraries were screened with DNA probes and specific antiserum. The open reading frame consisted of 663 nucleotides coding for a 221-amino acid (aa) polypeptide with a deduced Mr of 24,747. No obvious hydrophobic signal sequence, membrane-spanning domains, or potential N-glycosylation sites were found in this sequence. The identity of the clone and the accuracy of the sequence was verified by direct aa sequencing of the tryptic peptides derived from the purified rat liver enzyme. Primer extension analysis showed that the transcription start point of the rat liver COMT mRNA was 450 bp upstream from the translation start codon. A putative polyadenylation signal (ATTAAA) was found in the 3'-noncoding region. The predicted size of the COMT transcript was 1.8-2.0 kb, which could be confirmed from Northern hybridization analyses of the isolated rat liver mRNA. One polypeptide of 25 kDa, could be immunoprecipitated with anti-COMT antibody from in vitro translation of rat liver mRNA. Employing the DNA blot analysis only one COMT-encoding gene was found in the rat genome.