Cryopreservation of human hepatocytes is important for the treatment of liver disease by hepatocyte transplantation and also for the use of hepatocytes as an in vitro model of the liver. One factor in the success of cryopreservation is the quality of cells before freezing. Preincubation of hepatocytes with cytoprotective compounds to allow recovery from the isolation process prior to cryopreservation, such as those that will boost cellular adenosine triphosphate (ATP) content or antioxidants, may improve the viability and function of cells upon thawing. Rat hepatocytes were used to investigate the effects of preincubation with 10 compounds: precursors (glucose, fructose, glutathione, and S-adenosyl-L-methionine), antioxidants (ascorbic acid and α-lipoic acid), and compounds with multiple effects (N-acetylcysteine, pentoxifylline, prostaglandin E1, and tauroursodeoxycholic acid). Human hepatocytes were then used to investigate 5 of the original 10 compounds (glucose, fructose, α-lipoic acid, S-adenosyl-L-methionine, and pentoxifylline). Glucose preincubation (100 - 300 mM) improved the viability and attachment efficiency of rat hepatocytes and improved the viability and reduced lactate dehydrogenase (LDH) leakage of human hepatocytes. Fructose preincubation (100 - 300 mM) improved the viability and attachment efficiency of rat hepatocytes and improved the attachment efficiency of human hepatocytes. α-lipoic acid preincubation (0.5 - 5 mM) improved the viability and attachment efficiency of both rat and human hepatocytes. At a concentration of 2.5 mM α-lipoic acid also improved the albumin production of human hepatocytes. In conclusion, preincubation of hepatocytes prior to cryopreservation can improve the viability and function of thawed cells and may provide a method of obtaining better-quality cryopreserved hepatocytes for transplantation. Liver Transpl11:1533–1540, 2005. © 2005 AASLD.