The purpose of this investigation was to assess protection by zinc against acetaminophen induced hepatotoxicity and to evaluate possible mechanisms of protection. Mice were treated with zinc (3 mg/kg, ip) or saline (ip) 48 and 24 hr before and sacrificed 12 hr after acetaminophen administration (375, 500, or 750 mg/kg, po). Liver toxicity was then assessed by histological examination. The incidence of hepatotoxicity was significantly less at 375 and 500 mg/kg of acetaminophen in zinc treated animals. The same dosage of zinc was not hepatoprotective when given 1 hr after acetaminophen. Mice were also treated with 1 to 10 mg/kg of zinc (ip) 48 and 24 hr prior to sacrifice, and metallothionein, cytochrome P-450, glutathione, and UDP-glucuronosyl transferase (GT) were determined in the liver. Metallothionein and UDP-GT were increased and P-450 and glutathione decreased at the higher dosages of zinc; however, only metallothionein was significantly changed at the dosage of zinc (3 mg/kg) used in the hepatoprotection experiments. Further, mice were similarly treated with 3 mg/kg of zinc before administration of 375 mg/kg of [3H]acetaminophen (po) and the amount of acetaminophen and acetaminophen bound to metallothionein were determined in the liver for 0.5 to 24 hr. In addition, after 6 hr the subcellular distribution and covalent binding to protein of acetaminophen were also determined. Zinc treatment had no significant effect in any of the above determinations. These results indicate that zinc protects against acetaminophen induced hepatotoxicity and that the observed protection is probably due to an induced biochemical change, but it is apparently not the result of any of the commonly invoked mechanisms.