Aflatoxin B-1 (AFB(1)) is a fungal toxin that has been associated with primary hepatocellular carcinoma (HCC) in humans. This study was undertaken to determine the cellular and molecular mechanisms by which the antioxidants beta-carotene and lycopene inhibit AFB(1)-induced toxic changes in human hepatocytes (HepG2 cells). An in vitro system was optimized to test the chemoprotective effects of lycopene and beta-carotene on HepG2 cells exposed to different concentrations of AFB(1). Ultrastructurally, HepG2 cells cultured in the presence of AFB(1) showed mitochondrial damage, nuclear condensation and a loss of cell-to-cell contact; the latter was reflected in the observation of dysfunctional gap junctions, resulting in a loss of cell-to-cell communication. At the genomic level, AFB(1) formed AFB(1)-N7-guanine adducts, caused apoptotic cell death and suppressed p53 protein expression. In the presence of the carotenoids, survival of cells exposed to AFB(1) was increased, and there was also a significant increase in cellular mitochondrial activity. Our results demonstrate that HepG2 cells pretreated with lycopene and beta-carotene are protected from the toxic effects of AFB(1) at both the cellular and molecular levels.