An optimized computer-controlled freezing protocol for the cryopreservation of rat liver parenchymal cells was developed. The best survival rates were obtained when a slow cooling rate was used and when the supercooling was interrupted with a shock cooling to initiate ice nucleation. Ten percent dimethyl sulfoxide was added and removed gradually for best results. Thawed rat liver parenchymal cells had a viability, as judged by trypan blue exclusion, of 69% (SD = 6) versus 82% (SD = 7) for freshly isolated cells. The content and activities of the xenobiotic metabolizing enzymes, cytochrome P450, UDP-glucuronosyl transferase, and microsomal and cytosolic epoxide hydrolase, were not affected, whereas a slight reduction of glutathione S-transferase and sulfotransferase occurred. If cryopreserved cells were purified by a Percoll centrifugation after thawing the enzyme activities were not significantly different from those of freshly isolated parenchymal cells and also the viability was 86% (SD = 3). Cryopreserved rat liver parenchymal cells only metabolized about 50% of benzo(a)pyrene compared to freshly isolated cells. It is less likely that the reduction in enzyme activities was due to the cryopreservation procedure than that it was due to the loss of NADPH as a cofactor for cytochrome P450 which then resulted in the decreased xenobiotic metabolism. This cryopreservation protocol was also suitable for a variety of liver parenchymal cells from other species when trypan blue exclusion was used as a viability marker.