The successful use of isolated hepatocytes for transplantation will, no doubt, require cryopreservation of the cells. However, cryopreservation results in the loss of viability of isolated hepatocytes. In this study a method is described that allows recovery of viable hepatocytes after cryopreservation. Freshly prepared and cryopreserved hepatocytes (1.8 M Me2SO in Euro-Collins solution) were suspended in Dulbecco's culture medium and allowed to form monolayers in Primaria T-25 culture flasks. Viable hepatocytes produced monolayers after 3 h of incubation and viable cultures could be maintained for up to 7 days as judged by trypan blue exclusion. Metabolic viability was measured by incorporation of radiolabeled isoleucine into proteins. Maximal protein synthetic capabilities in freshly prepared hepatocytes was obtained 24 h after culturing. Cryopreserved hepatocytes showed a decrease in protein synthetic capabilities after 24 h culturing. However, longer times of culture resulted in regenerating the capacity of these cryopreserved cells to carry out protein synthesis to levels similar to those of freshly prepared and cultured cells. Thus, incubation of monolayer cultures of cryopreserved hepatocytes for at least 48 h provides a means for reversing the injury caused by freeze-thaw stress and the regeneration of initial viability. This technique may provide a method that is suited for the use of cryopreserved hepatocytes for clinical transplantation.