The present study was designed to prepare and characterize subcellular fractions from the liver of male C57B1/6 mice, with special emphasis on their suitability for use in studies of epoxide hydrolase isozymes. The effects of different washing and pelleting procedures on the mitochondrial, microsomal and cytosolic fractions were studied. It was found that 133,000 gav for 60 min (i.e. more extensive force than the usual 105,000 gav for 60 min) was necessary to obtain a membrane-free cytosolic fraction, while one wash for microsomes and two washes for mitochondria yielded reasonably pure fractions. The purity of the different fractions obtained by differential centrifugation was then determined using established enzyme markers and morphological examination with the electron microscope. Several enzymes involved in drug metabolism were also measured in these fractions. The subcellular distributions obtained here for marker enzymes closely resemble those reported for rat liver. Starvation had no significant effect on the epoxide hydrolase activities nor did the addition of mouse bile or rat liver cytosol, which might contain inhibitors. The change in epoxide hydrolase activities with time after preparation of the subcellular fractions was studied, as well as the effect of freeze-thawing. The subfractions prepared here are suitable for the further characterization of the different forms of epoxide hydrolase present in mouse liver, as well as for other studies requiring well-characterized subfractions.