We examined the response of murine oval cells, that is, the putative liver progenitor cells, to acetaminophen. Female C57BL/6J mice were injected intraperitoneally with varying doses of N-acetyl-paraaminophen (APAP) (250, 500, 750, and 1,000 mg/kg of weight) and sacrificed at 3, 6, 9, 24, and 48 hours. In preliminary studies, we showed that anticytokeratin antibodies detected A6-positive cells with a sensitivity and specificity of greater than 99%. The oval cell reaction was quantified, on immunostaining for biliary-type cytokeratins, as both number and density of oval cells per portal tract, analyzed by size of portal tract. Acetaminophen injury was followed by periportal oval cell accumulation displaying a moderate degree of morphological homogeneity. Oval cell response was biphasic, not temporally correlating with the single wave of injury seen histologically. Increases in oval cells were largely confined to the smallest portal tracts, in keeping with their primary derivation from the canals of Hering, and increased in a dose-dependent fashion. The timing of the two peaks of the oval cell reaction also changed with increasing dose, the first becoming earlier and the second later. In conclusion, our studies indicate a marked oval cell activation during the height of hepatic injury. Oval cells appear to be resistant to acetaminophen injury. The close fidelity of mechanism and histology of acetaminophen injury between mouse and human livers makes it a useful model for investigating liver regeneration and the participation of stem/progenitor cells in that process. (HEPATOLOGY 2005.)