Large-scale, high-density freezing of hybridomas was studied to apply frozen cells to start high-density culture. We showed here that hybridomas can be frozen at 1.5 × 108 cells/mL, without decrement in viability and proliferating activity. Blood transporting bags were used for large-scale freezing to store 25 mL of cell suspension with a cell density, 1.5 × 108/mL. The number of cells stored in a bag (3.0 × 109 cells) was enough to start a high-density culture at a 10 times higher cell density (6.0 × 106 cells/mL) than normal inoculation, and the cells proliferated to 107 cells/mL within 2 days. These results indicate that the large-scale freezing method is useful for large-scale culture of mammalian cells.