The relationship between the metabolic disposition of acetaminophen and the susceptibility of hamsters, mice and rats to acetaminophen-induced liver necrosis has been examined. The fraction of low doses of acetaminophen converted to the mercapturic acid metabolite was highest in the most susceptible species (hamsters, mice), and lowest in the more resistant species (rat). Pretreatment regimens known to potentiate the hepatotoxicity increased mercapturic acid formation whereas treatments which protect from liver damage decreased mercapturic acid formation. The data suggest that the activity of the mercapturic acid-forming pathway in vivo reflects the activity of the hepatotoxic pathway. As the dose of acetaminophen was increased to hepatotoxic levels, the fraction of the dose excreted as the mercapturic acid decreased markedly, commensurate with the known depletion of hepatic glutathione under these conditions. It is suggested that the normal metabolic intermediate which conjugates with glutathione in the mercapturic acid pathway is also the electrophilic metabolite which in the absence of glutathione arylates hepatic macromolecules and causes cell death.