The protective effect of a single dose of ethanol with regard to hepatotoxicity caused by acetaminophen (APAP) can be a consequence of either direct or indirect inhibition of APAP oxidation to its hepatotoxic intermediate (N-acetyl-p-benzoquinoneimine, NAPQI), or augmentation of repair mechanisms following the hepatotoxic insult. The mechanism of hepatoprotection appears to be species dependent. By varying the time of ethanol administration relative to APAP in mice (30 min before to 240 min after APAP), it was shown that ethanol must be administered early relative to APAP for hepatoprotection to be maximized. The role of direct inhibition of cytochrome P450 in the hepatoprotective effect of ethanol was evaluated by comparing the hepatoprotection afforded by ethanol and 4-methylpyrazole (4-MP) in vivo to the inhibition of APAP oxidation to NAPQI caused by each in mouse liver microsomes. At their respective peak in vivo concentrations attained following hepatoprotective doses, both ethanol and 4-MP inhibited the oxidation of APAP in microsomes by 25-30%. This result suggests that direct inhibition of cytochrome P450 by ethanol plays a role in the protection against acetaminophen-induced hepatotoxicity in mice. In human liver microsomes the inhibition of APAP oxidation to the hepatotoxic intermediate by 48 mM ethanol is less than half of the apparent inhibition of APAP oxidation reported in clinical studies in which the maximum ethanol concentration would have been 15-20 mM. Thus, in contrast to the mouse, inhibition of APAP oxidation to NAPQI in humans appears to be largely indirect, as has been reported previously in the rat.