Catechol-O-methyltransferase (COMT; E.C. 2.1.1.6) from human frontal cortex occurs in both a soluble and membrane-bound form. Attempts to solubilize the membrane-bound transferase by repeated washing or by extraction into solutions of high ionic strength were unsuccessful. The finding that Triton X-100 was capable of solubilizing membrane-bound COMT suggested that the membrane-bound transferase is an integral membrane protein. The membrane-bound and soluble enzymes did not differ in their requirements for magnesium ions or in their pH-activity profiles; both enzymes showed an optimum near pH 8.0 when assayed in phosphate buffer. In addition, the two enzymes did not differ in the degree of inhibition caused by CaCl2, both enzymes displaying 65% inhibition at 2.5 mM CaCl2. The competitive inhibitors tropolone and nordihydroguaiaretic acid displayed Ki values for the membrane-bound transferase five- to 10-fold lower than those observed for the soluble transferase. Solubilization of membrane-bound COMT in Triton X-100 resulted in an increase in the apparent Km value of the membrane-bound transferase for dopamine. The increase in Km appeared to be due to apparent competitive inhibition by Triton X-100 and reached a limiting value of approximately 80 microM. These results confirm that membrane-bound COMT is an integral membrane protein that may be structurally distinct from soluble COMT.