Accurate interpretation of microscopic changes in tissues is critical in hazard identification and risk assessment. To address a possible confounder, the effects of postmortem interval on hepatocyte vacuolation and liver weight were studied in fasted and nonfasted Sprague-Dawley rats. Male and female rats (5/sex/interval) were euthanized with CO2, weighed, and necropsied either immediately or after remaining in the closed CO2 chamber for 5, 10, or 25 minutes after respirations ceased. The liver was removed, weighed, and fixed for light microscopy, immunohistochemistry, and electron microscopy. The liver weight and liver to body weight ratio increased significantly in both male and female rats. Postmortem hepatocellular vacuolation was more prominent in males than in females. Both fasted and nonfasted males were similarly affected, however, fasted females were affected more than nonfasted females at the 25-minute interval. Ultrastructurally, intracytoplasmic vacuoles in hepatocytes and/or endothelial cells contained electron-lucent material that was morphologically similar to plasma in sinusoidal spaces. Results of our study suggest that hepatocyte vacuoles were formed in a postmortem time-dependent manner as a result of plasma influx into the cytoplasm. This change was associated with hepatic sinusoidal congestion and increases in liver weight. Males were more sensitive than females to postmortem hepatocyte vacuolation.