The biotransformation of etheofazine, a new anticancer drug, in hepatic subcellular fractions and its species differences were studied by determining the concentration of etheofazine, and its metabolite in hepatic 9000 x g supernatant fractions (S-9) and microsomes by HPLC.The in vitro liver intrinsic clearance of etheofazine in rat, dog and monkey liver S-9 was 14.9, 10.4 and 30.4 μL/min/mg protein, resp.The metabolite in hepatic microsome was 7-ethyl-8-aminotheophylline (EAT).There were insignificant species differences in metabolic profile of etheofazine.The results showed that hepatic subcellular fractions may be used as in vitro models for species difference study.