The hepatic toxic response to acetaminophen (APAP) is characterized by centrilobular (CL) necrosis preceded by hepatic microvascular injury and congestion. The present study was conducted to examine changes in liver microcirculation after APAP dosing. Male C57Bl/6 mice were treated with APAP (600 mg/kg body weight) by oral gavage. The livers of anesthetized mice were examined using established in vivomicroscopic methods at 0, 0.5, 1, 2, 4, 6, 12 hours after APAP. The levels of hepatic transaminases (i.e., alanine aminotransferase [ALT] and aspartate transaminase) increased minimally for up to 2 hours. Thereafter, their levels were significantly and progressively increased. The numbers of swollen sinusoidal endothelial cells (SECs) in periportal regions were increased (3.5-fold) from 0.5 to 6 hours, and those in CL regions were increased (4.0-fold) at 0.5 and 1 hour. The intensity of in vivostaining for formaldehyde-treated serum albumin, which is a specific ligand for SECs, was reduced from 2 to 12 hours. Erythrocytes infiltrated into the space of Disse as early as 2 hours, and the area occupied by these cells was markedly increased at 6 hours. Sinusoidal perfusion was reduced from 1 through 12 hours, with a nadir (35% decrease) at 4 and 6 hours. Phagocytic Kupffer cell activity was significantly elevated from 0.5 through 12 hours. Although gadolinium chloride minimized the changes in sinusoidal blood flow and reduced ALT levels 6 hours after APAP, it failed to inhibit endothelial swelling, extravasation of erythrocytes, and CL parenchymal necrosis. These results confirm that APAP-induced SEC injury precedes hepatocellular injury, supporting the hypothesis that SECs are an early and direct target for APAP toxicity. These findings also suggest that reduced sinusoidal perfusion and increased Kupffer cell activity contribute to the development of APAP-induced liver injury. Microcirculation(2003) 10,391–400. doi:10.1038/sj.mn.7800204