Phase I and phase II xenobiotic metabolism in fresh and cryopreserved precision-cut liver slices from the male rat was compared using a range of substrates. Liver slices were cryopreserved using a method according to de Kanter et al. (1998) with some modifications. This procedure involves incubation of the slices in 1-chloro-2,4-dinitrobenzene (DMSO) as a cryoprotector. The liver slice thickness was adjusted for an optimal maintained enzyme activity. The starting thickness of 250 µm appeared to be the best. An increased cryoprotectant concentration (from 12 to 18 % DMSO) enhanced phase II enzyme activity after cryopreservation of liver slices. Liver slices with 250 µm in thickness and a cryoprotectant concentration of 18 % DMSO was used throughout the study. Testosterone and ropivacaine were used as probe substrates to confirm previous results on phase I enzyme activities; a preserved metabolite formation in liver slices after cryopreservation. 7-hydroxycoumarin and 1-naphthol were used as probe substrates to study the maintenance of phase II metabolism after cryopreservation. The cryopreservation method used in this study resulted in maintained activities for both phase I and phase II xenobiotic metabolizing enzymes. This assay constitutes a cryopreservation method for male rat liver slices, good for both phase I and phase II metabolism with a significant improvement in liver slice applications. Liver slices with preserved enzyme activity can be a useful tool in drug metabolism studies, as they are easy to prepare and long-time storage is possible. A cryopreservation method of liver slices offers a more efficient use of available human material, since the access of human material is scarce and irregular.