In rats and baboons, the hepatotoxicity of chronic ethanol consumption is exacerbated by beta-carotene feeding, but the mechanism of this adverse effect is unknown. In this study, the toxicity of beta-carotene and acetaldehyde was documented by the MTT test (an assay of reduction of tetrazolium to formazan) and by lactate dehydrogenase (LDH) leakage. In HepG2 cells, beta-carotene or acetaldehyde inhibited mitochondrial reduction function as indicated by a decrease of the MTT test. beta-Carotene was inhibitory at very low concentration, in a dose-dependent manner. The combination of these two compounds resulted in an additive effect. Acetaldehyde increased LDH leakage from the HepG2 cells into the medium, whereas beta-carotene by itself did not show such an effect, but it exacerbated the toxicity of acetaldehyde when combined. In addition, this study showed that acetaldehyde and beta-carotene inhibited each other's clearance from the medium, which suggests that these two chemicals may share, at least in part, a common metabolic pathway (possibly via aldehyde dehydrogenase) in the cells, and that a competitive inhibition may exist. In conclusion, this preliminary study indicates that beta-carotene is toxic to hepatocytes, especially when combined with acetaldehyde, the metabolite of ethanol.