A radiochemical microassay for the determination of phenol O-methyltransferase (PMT) activity in human red blood cell membranes has been developed. Acetaminophen was used as the substrate. The apparent Michaelis-Menten (KM) value for acetaminophen was 21.2 X 10(-3) M. The apparent KM value for S-adenosyl-L-methionine, a co-substrate for the reaction, was 4.8 X 10(-6) M, and the pH optimum of the reaction was approximately 9.0 with four different buffer systems. Phenol was also tested as a substrate and had an apparent KM value of 2.0 X 10(-3) M. Human erythrocyte (RBC) membrane PMT activity did not have the biochemical characteristics of catechol O-methyltransferase, another RBC membrane methyltransferase enzyme activity. Blood samples obtained from 212 randomly selected adult white subjects had a mean activity of 134.5 +/- 41.5 pmol of p-acetanisidide formed per mg protein per hour (mean +/- S.D.). Activities varied from 44 to 282 units. There were no differences in the mean activities of samples from men and women. Experiments in which mixtures of "low" and "high" activity RBC membrane preparations were assayed for PMT provided no evidence that the variations in enzyme activity were due to the presence of endogenous PMT activators or inhibitors. RBC membrane PMT activity in blood from 9 patients with renal failure, a pathological state in which there are elevated circulating levels of phenols, was found to be significantly decreased with average activity of 76.2 +/- 9.7 (mean +/- S.E.M., P less than 0.001).