AbstractThe aim of this study was to define the long‐term stability of metabolizing enzymes in activating preparations for short‐term genotoxicity bioassays under various storage conditions. Expressions of cytochrome P450 content, NADPH‐cytochrome (P450) c‐re‐ductase activity, and of the several monooxygenases, such as aminopyrine N‐demethylase (class IIIA P450), p‐nitroanisole O‐demethylase (mixed), dinemorphan N‐demethylase (HB1), ethoxyresorufin O‐deethylase (IA1), ethoxycoumarin O‐deethylase (mixed), and pentoxyresorufin O‐dealkylase (IIB1), were examined in S9 fractions derived from Na‐phenobarbital (PB) plus β‐naphthoflavone (β‐NF) induced male and female mice, stored at −80°C, or lyophilized and stored at −20°C. Lipid peroxidation was also determined. Cytochrome P450 and the associated activities were decreased by 30–82% within 9 months of storage. The pattern and degree of relative stabilities were different for the various isoforms. The IA 1‐like activity, for example, was much more stable (˜49%loss) than IIB 1‐like activities (up to 82% loss). In general, lyophilized enzymes were less stable than directly frozen preparations. In addition, immediately after freeze‐drying (lyophilization), a marked decrease in activity of up to 35% was observed. On the contrary, demethylation of aminopyrine and p‐nitroanisole remains almost constant over 6 months storage at −196°C. The results obtained indicate that either fresh, daily made S9 fractions or, alternatively, fractions stored in liquid nitrogen (up to 6 months) are recommen