Hepatotoxic doses of acetaminophen cause early impairment of Ca²⁺ homeostasis in the liver. This in vivo study considers the nucleus as a possible site of lethal Ca²⁺ action by evaluating whether acetaminophen raises Ca²⁺ in this compartment, whether DNA becomes altered, and whether DNA changes occur early enough during injury to contribute causally to necrosis. Fed Swiss mice were treated with 600 mg/kg acetaminophen ip and livers and blood samples were collected over time. Total nuclear Ca²⁺ accumulation and fragmentation damage to DNA showed modest parallel increases between 2 and 6 hr, followed by > 200% rises at 12 hr mirroring the appearance of frank liver injury (ALT > 10,000 U/liter). However, agarose gel electrophoresis revealed extensive loss of large genomic DNA from 2 hr onward, accompanied by the appearance of periodic DNA fragments. Thus, acetaminophen raised nuclear Ca²⁺ concentrations and promoted DNA fragmentation in vivo. The considerable cleavage of DNA seen at late times probably resulted from cell death, whereas loss of large genomic DNA from 2 hr onward appeared at an early enough point in time to be a contributing factor in acetaminophen-induced liver necrosis.