This study describes the effects of three cryopreservation media on the specific activity of corneal endothelial plasma membrane Na+,K+ATPase activity, a transporter required for the fluid pump in the cornea. Bovine corneal endothelial cell cultures were used as a model system for these studies. Cryopreserved primary cells were thawed and passaged once to increase cell number. The specific activity plasma membrane Na+,K+ATPase activity was subsequently measured on 4–6 replicate cultures. One freeze/thaw cycle depleted the Na+,K+ATPase specific activity of corneal endothelial cell cultures by approximately 90%, as compared to cells of equivalent passage which had not been cryopreserved. Cell morphology of the cryopreserved cultures was indistinguishable from that of control cultures. In other experiments, first passage cultures which had not been subjected to cryopreservation were incubated with a dimethyl sulfoxide-, glycerol-, or propane diol-based freezing medium and Na+,K+ATPase was measured on plasma membranes subsequently isolated from the cultures. Incubation of cells with cryopreservation media in the absence of the freezing process also depleted Na+,K+ATPase by approximately 90%. Radiolabeled ouabain was used to measure Na+,K+ATPase sites on cell cultures pretreated with dimethyl sulfoxide-based freezing media. A 4 h treatment with DMSO-based freezing medium had no effect on ouabain binding; treatment for 18 h reduced binding by only 50%. Thus, the method used to assess pump function (determination of Na+,K+ATPase specific activity versus ouabain binding) may provide conflicting data concerning the level of pump function cultured cells. The cryoprotectants present in many common media used to freeze tissue culture cells appear to inhibit corneal endothelial Na+,K+ATPase. Since the fluid pump of corneal endothelial cells is coupled to Na+,K+ATPase activity, care must be taken to insure that pump function is not impaired during cryopreservation of cell cultures.