BACKGROUND/AIMS: The cryopreservation of primary hepatocytes could avoid unnecessary isolation of hepatocytes and meet repeated investigational demands. We tried to find out an optimal cryopreservation method of rat hepatocytes. METHODS: Primary hepatocytes with more than 90% viability were cryopreserved with the manual or computer-programmable freezing method. We analyzed the effects of the composition (basal medium or fetal bovine serum, FBS) of cryopreservation media, cell concentration, and freezing method on cell viability. RESULTS: Two-step addition of cryopreservation medium (4%--<16% DMSO) improved cell viability, compared to its one-step addition (82.7+/-2.5 vs 73.3+/-2.1%, p=0.008). In the manual method, the cell viability was about 60% and the culture attachment rate was less than 1%. They were not related with the composition of media used. These results showed that the manual method was not efficacious for cryopreservation. However, about 80% of the cell viability and 50% of the culture attachment rate could be obtained with an optimal computer-programmable method (-2C degrees slow cooling rate with a shock cooling, 2x106/mL cell concentration, 10-20% FBS, 10% DMSO). Moreover, the culture attachment rate was increased up to 75% when Percoll density purification was applied. CONCLUSIONS: Primary hepatocytes can be effectively cryopreserved by an optimal computer-programmable freezing method and Percoll density purification, but not by the manual method.