A partially purified preparation of hydroxy-indole-O-methyltransferase (HIOMT) from bovine pineal was shown to O-methylate estradiol-17beta (E2). The HIOMT preparation was incubated with E2 and [3H]-S-adenosylmethionine (SAM). The [3H]-3 methyl-ether or estradiol [MeO-E2] produced was identified by thin layer chromatography (TLC) and crystallization to constant 3H/14C ratio in the presence of [14C]-MeO-E2 and unlabeled MeO-E2 standards. The kinetics of the enzyme reaction for melatonin formation from N-acetyl-serotonin (NAS) and for MeO-E2 were compared.