Catechol-O-methyltransferase (COMT, EC 2.1.1.6) catalyzes the O-methylation of a wide array of catechol-containing substrates using s-adenosyl-L-methionine as the methyl donor. In the present study, we have cloned and expressed the human soluble and membrane-bound COMTs (S-COMT and MB-COMT, respectively) in Escherichia coli and have studied their biochemical characteristics for the O-methylation of representative classes of endogenous catechol substrates (catecholamines and catechol estrogens) as well as exogenous catechol substrates (bioflavonoids and tea catechins). Enzyme kinetic analyses showed that these two recombinant human COMTs are functionally active, with catalytic and kinetic properties nearly identical to those of crude or purified enzymes prepared from human tissues or cells. Kinetic parameters for the O-methylation of various substrates were characterized. In addition, computational modeling studies were conducted to better understand the molecular mechanisms for the different catalytic behaviors of human S- and MB-COMTs with respect to s-adenosyl-L-methionine, various substrates, and also the regioselectivity for the formation of mono-methyl ether products. Our modeling data showed that the binding energy values (Delta G) calculated for most substrates agreed well with the measured kinetic parameters. Also, our modeling data precisely predicted the regioselectivity for the O-methylation of these substrates at different hydroxyl groups, the predicted values matched nearly perfectly with the experimental data.