The cytotoxic effects of 3-tert-butyl-4-hydroxyanisole (BHA) and its metabolites, 3-tert-butylhydroquinone (tBHQ) and 3-tert-butyl-4,5-dihydroxyanisole (BHA-OH), were investigated in freshly isolated rat hepatocytes. These compounds caused a time-dependent cell death accompanied by loss of intracellular ATP, glutathione (GSH) and protein thiols at concentration of 0.5 mM. Supplementation of the hepatocyte suspension with 5 mM N-acetylcysteine, a precursor of intracellular GSH, significantly delayed the onset of cytotoxicity induced by BHA-OH and tBHQ; the loss of intracellular ATP, GSH and protein thiols was also prevented. Although N-acetylcysteine did not affect BHA disappearance in the cell suspension, disappearance of tBHQ and formation of tBHQ-GSH conjugate were stimulated by N-acetylcysteine. In addition, N-acetylcysteine prevented BHA-OH disappearance and 3-tert-butyl-5-methoxy-1,2-benzoquinone (BHA-Q) formation. In isolated hepatic mitochondria, BHA, tBHQ and BHA-OH impaired respiration related to oxidative phosphorylation; tert-butylquinone (tBQ) and BHA-Q, quinones derived from tBHQ and BHA-OH, resulted in the significant inhibition of mitochondrial respiration. These results indicate that BHA-OH is the most cytotoxic followed by tBHQ and BHA and that protein thiols and mitochondrial respiratory system are important targets for BHA and its intermediates.