1. Rat hepatocytes were cryopreserved using a number of procedures and the viability, attachment, and metabolic activity of the cryopreserved cells were compared to freshly isolated hepatocytes. Several cryopreservation agents (dimethylsulphoxide [DMSO], glycerol, polyvinylpyrrolidone [PVP], dextrans), and combinations of these agents, were examined. Other variables tested included the freezing rate, thawing rate, and the concentration of serum in the freezing medium. 2. Recovery of viable attached cells was optimal using DMSO at concentrations of 10% or higher, a slow stepwise cooling procedure, and a quick thaw. The concentration of serum in the freezing medium (0% to 90%) did not affect cryopreservation results. Using this procedure the recovery of viable hepatocytes was 70%. 3. Levels of hepatocyte ethoxycoumarin-O-deethylase (ECOD) activity did not change following cryopreservation. The rate of decline of ECOD activity with time in culture was similar in freshly isolated and cryopreserved hepatocytes. 4. Hepatocytes isolated from three human livers were cryopreserved and recovered with viabilities similar to those obtained with the rat. A preliminary experiment also showed no loss of metabolic activity in human hepatocytes following cryopreservation.