The development of a reproducible, effective system of cryopreservation of hepatocytes would create new possibilities for metabolic studies, as well as the clinical application of hepatocellular transplants in diverse hepatic disorders. For the purpose of dealing with both questions, we have studied different media and procedures of freezing, and have chosen that combination which afforded the best results for long-term studies of function. A series of intrasplenic transplants were performed with freshly isolated hepatocytes as well as cryopreserved ones, and several hepatospecific parameters (Alanine aminotransferase, ornithine carbamoyl transferase, lipogenesis, uptake of 99mTc-N-(p-butylphenylcarbamylmethyl)iminodiacetic acid, G6Pase, albumin, and gamma-glutamyl transpeptidase (gamma-GT) were quantified for 9 months. The histological study of the cells reveals that they acquired an architecture characteristic of the liver. All of the parameters indicative of hepatic function were detected throughout the 9-month period in the transplanted spleens. All the spleens, controls and transplanted, were found negative for the presence of the dedifferentiation marker gamma-GT. These results clearly indicate the possibility of recovering hepatocytes which maintain their specific functions after being subjected to a freezing-thawing process, with the corresponding implications for future clinical application as well as for hepatic biochemistry.