Melatonin, the major secretory product of the pineal gland, is in focus of many research areas because of its ability to scavenge free oxygen radicals and thereby protect cells and tissues from radical damage. Some studies suggest melatonin may be a possible therapeutic agent with potential clinical applications against pathological states due to reactive oxygen species. Here, we investigated the effects of melatonin on the mouse hepatoma cell line HEPA 1-6, coincubated with ethanol, and tamoxifen, respectively. Cell proliferation rates were detected by the 3-[4,5 dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide (MTT) proliferation assay. A dose-dependent inhibition of the proliferative activity by melatonin was observed from 640 microM to 3 mM, which was significantly higher (P < 0.01) than with the solvent (ethanol) alone. Concentrations of 320 microM and less had no effect on cell proliferation. This antiproliferative effect might be because of the prolonged activation of mitogen-activated protein kinase which was activated by phosphorylation 15 min after the induction with melatonin. Furthermore, apoptosis was found to be enhanced by melatonin (75% more than with the solvent alone, P < 0.001). Finally, we show that the inhibitory effect of tamoxifen (25 microM) is markedly enhanced by the coincubation with melatonin (1.3 mM) up to 75% (P < 0.001). These data show that the antiproliferative effects of tamoxifen and ethanol, respectively, on mouse hepatoma cell line HEPA 1-6 are enhanced by melatonin. Although at the conditions described here the antiproliferative effects of melatonin occur at supraphysiological concentrations, these data may help to support clinical studies where melatonin is given simultaneously with tamoxifen or other standard chemotherapeutica.