A satisfactory and reproducible technique of cryopreservation of canine lymphocytes for use in mixed leukocyte culture (MLC) and cell-mediated lympholysis tests has been developed. Dimethyl sulfoxide was used as the cryopreservation agent. Cells were frozen to -50 C at a controlled rate (-1 C/min) and stored at -169 C. The best preservation was obtained with a concentration of 10 x 10(6) lymphocytes/ml in Waymouth's MB-752/1 medium supplemented with 30% dog serum (MB-30). Before use in MLC or cell-mediated lympholysis tests, lymphocytes were rapidly thawed at 40 C and then rapidly diluted with MB-30 at room temperature. For best responses in MLC, one fresh component (either stimulating or stimulated cells or serum) was needed. The magnitudes of responses of frozen lymphocytes to phytohemagglutinin or in MLC were similar to those seen with fresh cells, but peak responses occurred usually 24 hr after those seen with fresh cells.