In the present study we evaluated viability and detoxifying enzyme capacity of cryopreserved hepatocytes from various species, including man, immobilized in calcium alginate gels. Ethoxyresorufin O-deethylase, phenacetin deethylase, pentoxyresorufin O-dealkylase, tolbutamide hydroxylase, S-mephenytoin hydroxylase, dextromethorphan demethylase, and nifedipine oxidation corresponding to the major cytochromes P450 (CYP) involved in xenobiotic metabolism as well as whole glutathione S-transferase (GST) activity were measured using specific substrates and after exposure or not to prototypical inducers. After deep-freeze storage, viability of immobilized hepatocytes was only slightly reduced and most CYP-related monooxygenase activities were well preserved, being expressed at levels close to those measured in unfrozen hepatocyte monolayers. By contrast, total GST activity was decreased by around 50%. However, as did CYP1A- and 3A-related enzymes, rat GST remained capable of responding to prototypical inducers. The fold increases were comparable in unfrozen and frozen immobilized hepatocytes and in unfrozen hepatocyte monolayers. The duration of storage, even when exceeding one year, did not affect viability and functions. In conclusion, after cryopreservation, alginate-entrapped hepatocytes remain highly viable and capable of expressing most detoxifying enzymes at levels close to those expressed in corresponding unfrozen hepatocyte monolayers and of responding to prototypical inducers.