The experiment aimed at studying long-term cryopreservation of hepatocytes and therapeutic effect of their transplantation for acute hepatic failure. Hepatocytes of wistar rats were cryopreserved in liquid nitrogen for 30, 60, 90, and 180 days. Their viabilities were respectively 71.2%, 75.7%, 69.7%, and 73.5% of fresh cells', however, there were no significant differences among all cryopreserved groups. Differences of viably intra-hepatocellular enzyme contents among all groups, such as glutamic oxaloacetic transaminase and so on, were not significant. Morphologically intact hepatocytes were observed under light microscopy and transmission electron microscopy. Survival rate of Sprague-Dawley rats with acute hepatic failure were increased from 1/8 to 5/7 because of the transplantation of syngeneic frozen hepatocytes (P = 0.035). The results showed that after 30 days of cryopreservation in liquid nitrogen, the viability of hepatocytes decreased significantly, but no longer decreased in subsequent freeze up to 180 days. The viable intact cells and their normal enzyme contents were kept. The acute hepatic failure could be treated by them.