Long-term preservation of hepatocytes is essential to a wide variety of applications, including drug metabolism studies, cell transplantation, and extra-corporeal liver support. Our previous studies have shown that the pre-culture of hepatocytes prior to cryopreservation can enhance post-thaw viability. The aim of this study was to characterize the post-thaw function of the hepatocytes for longer periods of time and to quantify the role of post-thaw apoptosis in the results observed. To that end, hepatocytes were frozen, thawed, and cultured in a hollow-fiber bioreactor. Albumin secretion and oxygen consumption were measured over a period of up to 9 days. The post-thaw albumin secretion for cells pre-cultured for 24 h prior to cryopreservation was greater than that measured in cells that were not pre-cultured prior to cryopreservation. Annexin V-FITC/Propidium iodine staining and caspase-3 activity measurements indicated that apoptosis was higher in frozen and thawed cells that had not been pre-cultured compared to cells cultured to form aggregates. These results suggest that differences in post-thaw function for cells, with or without pre-culture, may involve differences in the propensity of post-thaw apoptosis.