Human lymphocytes can be cryopreserved for the purpose of performing in vitro tests of cellular immunity. A systematic study of the conditions for freezing and recovering cells has shown that there is wide flexibility in cell concentrations of cryoprotective agent and the temperature and rate of dilution however, have definable optima. Cells frozen and thawed under optimal conditions retain their reactivity in MLC and can be used for sequential studies of immune responsiveness. Approximately 70% of viable and functional cells are recovered when the cells are frozen in 7.5--12.5% DMSO and no further cryoprotection is discernable when up to 50% serum is added to the freezing media. The temperature and rate of dilution are critical only in that cells diluted rapidly in THE COLD (10-fold in 2 min at 0 degrees C) are less responsive in MLC than are cells diluted slowly (10-fold in 10 min) in the cold or slowly or rapidly at room temperature.