Catechol-O-methyltransferase (COMT) is an enzyme that plays a key role in the modulation of catechol-dependent functions such as cognition, cardiovascular function, and pain processing. Recently, our group demonstrated that three common haplotypes of the human COMT gene, divergent in two synonymous and one nonsynonymous position, are associated with experimental pain sensitivity and onset of temporomandibular joint disorder. In order to determine the functional mechanisms whereby these haplotypes contribute to pain processing, a series of in vitro experiments were performed. Haplotypes divergent in synonymous changes exhibited the largest difference in COMT enzymatic activity because of reduced amount of translated protein. The major COMT haplotypes varied significantly with respect to mRNA local stem-loop structures such that the most stable structure was associated with the lowest protein levels and enzymatic activity. Site-directed mutagenesis that eliminated the stable structure restored the amount of translated protein. These data provide the first demonstration that combinations of commonly observed alleles in the coding region of the human COMT gene can significantly affect the secondary structure of corresponding mRNA transcripts, which in turn leads to dramatic alterations in the translation efficiency of enzyme crucial for a variety of essential functions. The protocols applied to the study of these molecular genetic mechanisms are detailed herein.