【Objective】To prepare and primarily evaluate a hepatocyte—SV40LT antigen gene immortalized hepatocyte.【Methods】SV40LT antigen gene was transfected into the primarily cultured rat hepatocyte to get in vitro immortalized hepatocyte.We assessed its in vitro growth properties by drawing growth curve,and evaluated the cryopreservation method and compared with primarily cultured hepatocyte and liver tumor cell line CBRH7919.【Results】Primarily cultured rat hepatocyte could proliferate in vitro after SV40LT antigen gene transfection.SV40LT antigen gene transfected hepatocyte grew much more faster than the primarily cultured hepatocyte in vitro(P 0.05),slower than tumor cell(P 0.05).(74±4)% of the cryopreserved SV40LT antigen gene immortalized hepatocyte could be recovered,similar(P 0.05) to that from recovered primarily cultured hepatocyte(72±6)%.【Conclusion】SV40LT antigen gene transfected hepatocyte is in vitro immortalized.The recovery rate of the cryopreserved immortalized hepatocyte was good.These characters enable SV40LT antigen gene transfected hepatocyte an ideal sauce of hepatocyte.