This study examined whether the high sensitivity of plasma free metanephrines for diagnosis of pheochromocytoma may result from production of free metanephrines within tumors. Presence in pheochromocytomas of catechol-O-methyltransferase (COMT), the enzyme responsible for conversion of catecholamines to metanephrines, was confirmed by Western blot analysis, enzyme assay, and immunohistochemistry. Western blot analysis and enzyme assay indicated that membrane-bound and not soluble COMT was the predominant form of the enzyme in pheochromocytoma. Immunohistochemistry revealed colocalization of COMT in the same chromaffin cells where catecholamines are translocated into storage vesicles by the vesicular monoamine transporter. Levels of free metanephrines in pheochromocytoma over 10,000 times higher than plasma concentrations in the same patients before removal of tumors indicated production of metanephrines within tumors. Comparisons of the production of metanephrines in patients with pheochromocytoma with production from catecholamines released or infused into the circulation indicated that more than 93% of the consistently elevated levels of circulating free metanephrines in patients with pheochromocytoma are derived from metabolism before and not after release of catecholamines into the circulation. The data indicate that the elevated plasma levels of free metanephrines in patients with pheochromocytoma are derived from catecholamines produced and metabolized within tumors. Some tumors do not secrete catecholamines, but all appear to metabolize catecholamines to free metanephrines, thus explaining the better sensitivity of plasma free metanephrines over other tests for diagnosis of pheochromocytoma.