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Catechol-O-methyltransferase (COMT) is a key enzyme for inactivation and metabolism of catechols, including dopamine, norepinephrine, caffeine, and estrogens. It plays an important role in cognition, arousal, pain sensitivity, and stress reactivity in humans and in animal models. The human COMT gene is associated with a diverse spectrum of human behaviors and diseases from cognition and psychiatric disorders to chronic pain and cancer. There are two major forms of COMT proteins, membrane-bound (MB) COMT and soluble (S) COMT. MB-COMT is the main form in the brain. The cellular distribution of MB-COMT in cortical neurons remains unclear and the orientation of MB-COMT on the cellular membrane is controversial. In this study, we demonstrate that MB-COMT is located in the cell body and in axons and dendrites of rat cortical neurons. Analyses of MB-COMT orientation with computer simulation, flow cytometry and a cell surface enzyme assay reveal that the C-terminal catalytic domain of MB-COMT is in the extracellular space, which suggests that MB-COMT can inactivate synaptic and extrasynaptic dopamine on the surface of presynaptic and postsynaptic neurons. Finally, we show that the COMT inhibitor tolcapone induces cell death via the mechanism of apoptosis, and its cytotoxicity is dependent on dosage and correlated with COMT Val/Met genotypes in human lymphoblastoid cells. These results suggest that MB-COMT specific inhibitors can be developed and that tolcapone may be less hazardous at low doses and in specific genetic backgrounds.