The mechanisms of toxic effects of divalent cations of three heavy metals Hg, Cu and Cd in rat ascites hepatoma AS-30D cells cultivated in vitro were studied. In a recent publication (Belyaeva EA et al (2006). Biochim Biophys Acta 1757, 1568-1574), we have shown that in these cells, Cd2+ induces cell death, both necrotic and apoptotic, that is accompanied by increased generation of reactive oxygen species (ROS), most likely at the level of complex III of the respiratory chain, and opening of mitochondrial permeability transition pore. In the present investigation, we compared the effects of Cd2+ with those of Hg2+ and Cu2+ using the same cells. Changes in intracellular ROS formation and mitochondrial dysfunction were found to be the key events in heavy metal action on AS-30D cells. An increase of intracellular ROS production was an early and sustained phenomenon observed after Cu2+ treatment that was not followed by the dissipation of mitochondrial transmembrane potential. In the case of Hg2+ and Cd2+, ROS generation changes (an increase or decrease compared to control) were dose- and time-dependent as well; however, they were accompanied by the loss of the mitochondrial transmembrane potential. Among the metals tested, the most toxic was Hg2+, which induced both necrosis and apoptosis in a time- and dose-dependent fashion, whereas Cu2+ was weakly toxic and under the experimental conditions did not induce apoptotic death. We showed that cellular respiratory capacity was disturbed by all these heavy metals. In particular, Cu2+ induced stimulation of steady-state respiration rate, whereas Cd2+ decreased steady-state and CCCP-uncoupled cell respiration. High Hg2+ produced practically complete inhibition of the cellular respiration already at early stage of incubation. The data agree well with our observations on isolated mitochondria, where all these heavy metal ions produced severe changes of mitochondrial function manifested, though in different ways, in respiratory disturbance, pyridine nucleotide oxidation, changes of mitochondrial transmembrane potential and ROS production, mitochondrial swelling and K+ or/and Ca2+ release.