Previous results in Sprague‐Dawley rats indicate that acetone (A), methyl ethyl ketone (MEK), and methyl isobutyl ketone (MiBK) pretreatment (3 d, po) at dosages of 6.8 and 13.6 mmol/kg potentiate CCl4hepatotoxicity and CHCl3nephrotoxicity, respectively. The potentiation potency profile observed was MiBK > A > MEK for liver and A > MEK ≥ MiBK for kidney toxicity (Raymond & Plaa, 1995). In the present study, hepatic and renal microsomes from A‐, MEK‐, and MiBK‐pretreated rats (6.8 or 13.6 mmol/kg) were examined for cytochrome P‐450 content, substrate‐specific monooxygenase activity (aminopyrine and benzphetamine N‐demethylase, aniline hydroxylase) and in vitro covalent binding of14CHCl3and14CCl4. Of the three ketones, only MiBK significantly increased P‐450 content of liver and renal cortical microsomes. Similarly,14CCl4covalent binding under aerobic and anaerobic conditions was significantly increased by MiBK pretreatment only.14CHCl3covalent binding by renal cortical microsomes was significantly increased only under aerobic conditions by MiBK pretreatment. MiBK (13.6 mmol/kg) increased (threefold) aminopyrine N‐demethylation in both liver and kidney, but only benzphetamine N‐demethylation (twofold, at 6.8 and 13.6 mmol/kg) in liver; A and MEK had no effect on either monooxygenase. All ketones at dosages of 6.8 and 13.6 mmol/kg increased aniline hydroxylation in liver (two‐fold) and kidney (fivefold). Comparable profiles for P‐450 induction, haloalkane covalent binding, and aminopyrine or benzphetamine N‐demethylase activity were observed in liver and kidney microsomes. This profile was consistent with the ketone potentiation potency ranking profile observed in vivo for liver but not kidney injury. These findings affirm the importance of ketone‐enhanced bioactivation for potentiation of CCl4hepatotoxicity but suggest an alternative mechanism for CHCl3nephrotoxicity.