Dexamethasone (DEXA) administration has been associated with serum alanine aminotransferase (ALT) elevations that may result from enhanced ALT expression. The aim of our current study was to compare liver vs. serum ALT activity and to examine the onset of any hepatocellular changes. Groups of 4 male Sprague-Dawley rats were administered a single dose of DEXA or corn oil at 12, 16, and 24 h prior to euthanasia or once-daily for 2, 3, or 4 days. All (nonfasted) rats were necropsied together on Day 5. While DEXA incrementally increased liver ALT activity in the 1-, 2-, 3-, and 4-day treatment groups (maximal, 3.7-fold), liver aspartate aminotransferase (AST) never exceeded 1.4-fold over control. Significant hepatic glycogen elevations were detected after DEXA treatment, which correlated with microscopic observations. Serum ALT, AST, sorbitol dehydrogenase, and glutamate dehydrogenase (GLDH) increased after 2, 3, and 4 days of DEXA dosing (1.3-10.3-fold). DEXA-related necropsy findings included pale livers consistent with glycogen deposition. The relative percent liver to body weight was elevated in all DEXA-treated rats. Hepatocellular necrosis was observed in 1/4 rats at 12 h, 2/4 rats at 2 days, 4/4 rats at 3 days, and 3/4 rats at 4 days. DEXA treatment <2 days failed to produce consistent evidence of hepatic injury, as detected by serum biomarkers and pathology assessment. However, early DEXA treatment did correlate with apparent ALT induction. Ultimately, this may explain some early asymptomatic serum ALT elevations seen clinically.