We sought to evaluate the viability and function of cryopreserved hepatocyte spheroids using different cryopreservation solutions in order to elucidate the efficiency of cryopreservation. Hepatocytes isolated from a Sprague-Dawley rat were formed into spheroids by 24 hours of rotational culture. The spheroids were cryopreserved using a programmed linear freezer in a liquid nitrogen tank for 24 hours in four different cryopreservation solutions: UW solution (UW), William E media (WE), fetal bovine serum (FBS), and a mixture (MIX). After thawing, they were cultured for 4 days. With each hepatocyte spheroid, the viability using the MTT assay and hepatocyte-specific functions, such as ammonia clearance, urea nitrogen synthesis, and albumin secretion, were analyzed. The viabilities of cryopreserved hepatocyte spheroids were 64.8% +/- 10.2% (UW), 33.2% +/- 9.7% (WE), 69.3% +/- 8.7% (FBS), and 48.4% +/- 15.5% (MIX). Ammonia clearance of spheroids cyropreserved in UW solution was 0.93 +/- 0.13 mmol/L per well per day, which was not significantly different from freshly cultured spheroids. From the aspect of urea nitrogen synthesis, spheroids cryopreserved with UW, FBS, and MIX solution were not significantly different from freshly cultured spheroids. The amount of albumin secretion by the UW cryopreserved spheroids was significantly greater than that of other cryopreserved spheroids. Cryopreserved hepatocyte spheroids in UW solution were not significantly different from freshly cultured spheroids with respect to viability and function. UW solution was superior to other cryopreservation solutions for viability and functions.