To examine a role for disturbances in intracellular calcium homeostasis in acetaminophen-induced hepatotoxicity, freshly isolated mouse hepatocytes were incubated with 1.0 mM acetaminophen for 1.5 h to allow for covalent binding and initiation of cell damage. The hepatocytes were then washed and the cells incubated in fresh medium containing either 2.0 mM N-acetylcysteine or 1.5 mM dithiothreitol for the duration of a 4-h incubation period. These agents were used as tools in the elucidation of the biochemical events responsible for acetaminophen-induced cell necrosis. The reduced protein sulfhydryl content, cytosolic [Ca2+], and plasma membrane integrity were quantitated. Acetaminophen produced protein sulfhydryl depletion, an increased cytosolic [Ca2+], and cell injury; however, cytotoxicity preceded the increase in [Ca2+]. Both N-acetylcysteine and dithiothreitol restored the acetaminophen-induced protein sulfhydryl loss. Dithiothreitol prevented both further cell injury and an increase in the cytosolic [Ca2+]. However, cell death and a subsequent increase in cytosolic [Ca2+] proceeded unabated following N-acetylcysteine addition. Although both agents restored protein sulfhydryl content, in view of their contrasting ultimate effects on cell viability the role of reduced protein sulfhydryl depletion in acetaminophen-induced hepatic injury requires further investigation. The increase in cytosolic [Ca2+] with acetaminophen alone and with subsequent N-acetylcysteine addition was determined to be a secondary event in cell injury because cytotoxicity occurred by 1.5 h; however, the increase in cytosolic [Ca2+] was not observed until 2.5 h. Additional evidence for changes in cytosolic [Ca2+] as a secondary event was obtained by incubating the hepatocytes with acetaminophen in the presence of fura 2.(ABSTRACT TRUNCATED AT 250 WORDS)