Abstract Enzymatic O-methylation of catechol estrogens in red blood cells has been investigated with respect to species difference. In the presence of S-adenosylmethionine, 2- or 4-hydroxyestradiol (2-OHE 2 or 4-OHE 2) was incubated with blood lysate obtained from rats (five strains), guinea pigs, mice, rabbits, dogs, monkeys, and humans, respectively. The yielded guaiacols and unchanged substrate were determined by gas chromatography/mass spectrometry in a selected ion monitoring mode employing the corresponding 2H 4-labeled compounds as internal standards. The total amounts of guaiacols formed from 2-OHE 2 and 4-OHE 2 were different, being the highest (79.6% and 38.1%) in monkeys and the lowest (5.1% and 1.9%) in humans. The ratios of isomeric guaiacols formed from 4-OHE 2 (4Me/3Me) were 7.6–71, while those from 2-OHE 2 (2Me/3Me) were 1.4–3.2. Thus, marked differences in O-methylation of catechol estrogens were observed among animal species, but not significant strain difference was detected in rats.