Recent studies performed in our laboratory have shown that a brief period of preculture prior to cryopreservation improves the postthaw viability of hepatocytes. The purpose of this investigation is to characterize specific metabolic and biochemical characteristics of the hepatocytes (both frozen and nonfrozen) to help elucidate the role of preculture on the postthaw viability. Fresh and thawed hepatocytes were cultured in a bioartificial liver (BAL) to determine albumin secretion as a function of time in culture. In addition, cell extracts were analyzed using nuclear magnetic resonance (NMR) spectroscopy to quantify changes in cell membrane composition and energetics as a function of time in culture prefreeze and postthaw. The results of these studies showed an increase in albumin concentration in the culture medium with time in culture for the period tested for both fresh and frozen and thawed hepatocytes. NMR spectroscopy of lipid extracts indicates that in vitro culture of hepatocytes results in an increase in cholesterol relative to membrane phospholipid. Moreover, the NMR results also indicate phospholipid interconversion, via specific lipases in cultured hepatocytes, and these changes are consistent with water permeability measurements performed previously. Significant changes in phosphoenergetics were also observed, with the net energy charge for the cells increasing significantly with time in culture. In addition, NMR spectra show increased levels of g-phosphogluconate, another indicator of the cellular response to the stresses of isolation and ex vivo culture. These results suggest that energetic considerations may be a significant factor in the ability of hepatocytes to survive the stresses of freezing and thawing. Significant shifts in membrane phospholipids may also influence membrane permeability and postthaw survival.