Alpha-asarone (1) is a naturally occurring phenylpropene found in several plants, e.g. Acorus calamus. 1-containing plant materials and essential oils thereof are used for flavoring foods and in many phytopharmaceuticals. 1 has been claimed to have positive pharmacological effects, however, it is carcinogenic in male mice (liver) and probably genotoxic. Since the metabolic pathways of 1 have not been investigated and its carcinogenic mode of action is unknown, we investigated the metabolism of 1 in liver microsomes of rat, bovine, porcine, and human origin using HPLC-DAD and LC-ESI-MS/MS and derived kinetic data on the metabolite formation. The main metabolic pathway was the side-chain hydroxylation leading to (E)-3'-hydroxyasarone (2). Epoxidation of 1 presumably led to (E)-asarone-1',2'-epoxide (4) which instantly hydrolyzed to form erythro- and threo-configured diols (5b+5a). As a minor reaction O-demethylation of 1 was observed. The metabolite formation showed little species-specific differences with the exception of porcine liver microsomes for which the formation of diols 5b+5a exceeded the formation of alcohol 2. The kinetic parameters imply a dependence of the pattern of metabolite formation from substrate concentration. On the basis of our results and earlier findings we hypothesize the genotoxic epoxide 4 being the ultimate carcinogen metabolically formed from 1.