an assay for human erythrocyte catecholomethyltransferase activity using a catechol estrogen as the substrate a radiometric assay for catecholomethyltransferase comt activity in human erythrocytes is described that employs 2hydroxy3hestrone and nonradiolabeled sadenosylmethionine sam as the cosubstrates the ease of separation of the product of the reaction 2methoxy3hestrone from 2hydroxy3hestrone makes it possible to achieve low reaction blanks the assay is very sensitive and only 200 microliter of whole blood are used per determination the assay is highly reproducible the interassay variability coefficient of variation was 65 for 24 assays of comt activity in red blood cells in blood obtained daily for 24 days from one person in incubations conducted at 37 degrees c for 30 min the catecholomethyltransferase activity was a linear function of enzyme concentration equivalent to 11 to 180 microliter of packed red blood cells employing this assay we evaluated the catalytic conversion of 2hydroxyestrone to 2methoxyestrone by catecholomethyltransferase from human red blood cells and found that the apparent michaelis constant and the apparent maximal rate of reaction were 3 x 107 m and 67 x 109 mol ml1 erythrocytes h1 respectively the catecholomethyltransferase activity measured in erythrocytes obtained from 100 healthy subjects men and nonpregnant women was 82 017 mean se nmol 2methoxyestrone ml1 erythrocytes h1