Isolated human hepatocytes offer a unique way of studying the metabolism and mechanisms of action of drugs and toxic chemicals. Because of the irregular availability of human liver, a way of storing the hepatocytes until they can be conveniently used is required. Using rat and dog isolated hepatocytes, we have developed a procedure for cryopreserving hepatocytes in large numbers such as are needed for metabolism and toxicity studies. Hepatocytes were frozen in medium containing 10% dimethyl sulfoxide using a microcomputer-controlled freezing gradient and stored at -196 degrees C. Upon thawing, the total cell recovery for rat hepatocytes was 67%. Cell viability measured by trypan blue (TB) exclusion was 67%, 7-ethoxycoumarin (7-EOC) dealkylation 33%, and cytochrome P-450 75%, compared to fresh hepatocytes. With cryopreserved dog hepatocytes, the total cell recovery was 75%. TB exclusion was 62%, 7-EOC dealkylation 37%, and cytochrome P-450 68%, compared to fresh hepatocytes. The viability of cryopreserved hepatocyte preparations could be improved by density separation on Percoll giving a TB exclusion for rat hepatocytes of 85%, and 7-EOC dealkylation of 69% compared to fresh hepatocytes, with 67% of the viable cells recovered. Biphenyl was used as a substrate to measure integrated xenobiotic metabolizing activity by the hepatocytes. Total hydroxybiphenyl (OHB) formation, a mixed function oxygenase activity, was maintained in cryopreserved Percoll-treated rat hepatocytes at 86%, OHB glucuronide formation at 85%, and OHB sulfate formation at 20% of the values in fresh hepatocytes. In cryopreserved dog hepatocyte, total OHB formation was 39%, and OHB glucuronide and sulfate formation less than 10% of the values in fresh hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)