Summary Two tissue culture cell lines, HeLa and WI-38, were stored in the presence of 5% DMSO or 5% glycerol for intervals of 24 to 168 hrs at 4 and 37°C to test the toxic effects of these additives on the cells. The studies demonstrated the superiority of storage at 4°C. Viability was reduced after 72 hrs of storage of HeLa cells at 4°C and 24 hrs at 4°C was found to be the maximal storage time without toxic effects. During this time interval no demonstrable loss of viability was observed as monitored by the cell permeability and cell adhesion tests in either the frozen or unfrozen cells. Cell damage of the more delicate WI-38 cells was observed after 24 hrs at 4°C in both the prefreeze and post-thaw cultures; 6 hrs was the maximal storage period at 4°C without loss of viability. We recommend from the results of these studies that the cell lines be stored at 4°C when they cannot be immediately frozen after ampulizing. It is suggested that the more delicate diploid and primary cell cultures must be frozen within 3 to 6 hrs of ampulizing. Although the more hardy established cell lines may be left overnight at 4°C and successfully frozen on the next day with little loss in viability, it is advisable to freeze them within several hours after ampulizing to minimize cell damage.