Abstract: Objective To compare the growth and proliferative status of rat lymphocytes in different media, and to screen the new medium for lymphocyte culture.Methods Isolated and cultured rat thymus lymphocytes were cultured in a new medium, serum-free RPMI-1640 medium and a new serum-free medium.The growth states of the cells were observed with inverted microscope.The cell proliferative activity was detected by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT)colorimetric assay.Results Twenty-four hours after the inoculation, a large number of cells in different media adhered to the wall and spread evenly.Five days after the inoculation the number of cell growth was reduced, and the cells were declined.The new medium containing 0.1 mmol of methionine-enkephalin(MEK)could improve lymphocyte cell growth greatly.There was a significant difference in the cell proliferative activity between the new medium and traditional RPMI-1640 medium(P<0.05).The lymphocyte proliferation activity in RPMI-1640, the new medium(formulation 2)with serum, and the serum-free new medium(formulation 2)was 99.9%, 103.1%, and 96.6%, respectively.Conclusion The new medium prepared is more suitable for the growth and proliferation of rat lymphocyte.