ObjectiveTo explore the optimal methods of isolation and culture of hepatocytes from grass carp and serve the studies on nutritional metabolism and injury mechanisms in carp hepatocytes. MethodsProliferation of hepatocytes was tested by MTT assay. Function of THE hepatocytes was examined by measuring the levels of lactic acid dehydrogenase (LDH), albumin (ALB), and urea nitrogen (BUN) in the supernatant at different times, respectively. Results Using 0.25% warm trypsin digestion for 20 minutes each time, and to collect the hepatocytes step by step, the results of trypan blue test and blood cell counting chamber method showed that more than 99% of viable hepatocytes were obtained. ConclusionThe cells were cultivated in M199 medium supplemented with 10% fetal bovine serum and 10μg/mL insulin, at 7×10⁶ cell/mL concentration under the temperature of 28℃, 4.5% CO₂ for a long term. Primary culture of carp hepatocytes can be successfully obtained by the following procedure:cells cultured in M199 medium supplemented with 10% fetal bovine serum and 10 μg/mL insulin, at 7×10⁶ cell/mL concentration, under the temperature of 27℃ and 4.5% CO₂.