In the rat, the catechol O-methyltransferase (COMT) gene has been found to contain two promoters, P1 and P2. This organization enables the gene to produce a soluble (S-COMT) and a membrane-associated (MB-COMT) protein by using two in-frame ATG initiation codons (S- and MB-ATG). The P1 promoter expresses a 1.6 kb transcript (S-mRNA) which codes for the S-COMT polypeptide only. Here we demonstrate that the P2 promoter controls the expression of alternatively spliced 1.9 kb transcripts (MB-mRNA) which differ by a 27-nucleotide region immediately upstream of the MB-AUG codon. The presence of the 27-base sequence alters the nucleotide at position -3 from G to C, thereby changing the translation initiation context of the MB-AUG codon. Expression experiments in COS-7 cells using full-length COMT cDNAs showed that this alteration affected the initiation of the translation of the MB-AUG and consequently changed the relative amounts of MB- and S-COMT polypeptides produced. No proteolytic cleavage of the MB-COMT form to S-COMT was detected in in vitro or in vivo pulse-chase experiments. We conclude that the bifunctional 1.9 kb mRNAs are able to produce both S-COMT and MB-COMT polypeptide by the leaky scanning mechanism of translation initiation.