Chronic alcohol consumption depletes hepatic vitamin A stores. However, vitamin A supplementation is hepatotoxic, which is further potentiated by concomitant alcohol consumption. It was suggested that polar retinol metabolites generated by alcohol-inducible cytochrome P4502E1 aggravate liver damage. However, experimental evidence supporting this hypothesis is lacking. To elucidate the effects of polar retinol metabolites on cultured HepG2 cells and primary rat hepatocytes, polar retinol metabolites were extracted from liver tissues of rats fed either an alcoholic or isocaloric control Lieber-DeCarli diet. Cell toxicity assays included morphology assessment, trypan blue exclusion test, and LDH/AST leakage. Staining for DAPI and acridine orange, FACS analysis, and Western blot for cleaved caspase-9 and -3 were used to detect apoptosis. Polar retinol metabolites caused marked cytotoxicity in a concentration- and time-dependent manner in both cell types reflected by morphological changes, a dramatic increase in trypan blue positive cells, and LDH/AST leakage. Toxicity was due to apoptosis, as demonstrated by a time-dependent increase of sub-G1 cellular events, a rapid loss of mitochondrial membrane potential, and a time-dependent activation of caspase-9 and -3. No toxicity was found with equivalent doses of the control extract from nonalcoholic rats. We demonstrate that polar retinol metabolites cause marked hepatocyte death through the induction of apoptosis.