Abstract: AIM: To establish a method for adult rat hepatocyte preparation and incubation and study the biotransformation of etheofazine, a new anticancer drug, in adult rat hepatocyte suspensions. METHODS: The concentration of etheofazine and its metabolite in hepatocyte suspensions was determined by HPLC method. In addition, the viability of adult rat hepatocyte was judged by the trypan blue exclusion test. Lactate dehydrogenase release and adenine nucleotide level were assessed. RESULTS: The metabolite in hepatocytes is 7 ethyl 8 aminotheophylline(EAT) and the result is in consistence with result obtained in isolated perfused rat liver.V_max=12 80 nmol·min^-1/10⁶ cells,Km=640.9 μmol·L^-1 , liver intrinsic clearance CL_int=20.0μL·min^-1/10⁶cells. CONCLUSION: Hepatocyte culture is shown to be a good in vitro model for drug biotransformation study and etheofazine can decrease the cellular energy charge in hepatocyte suspensions.