Abstract: Aims/Background: Hepatocellular damage has been reported as a consequence of 3,4-methylenedioxymethamphetamine (MDMA) intake. However, little is known about the cellular mechanisms involved. The present study was undertaken to evaluate the effects of MDMA on cell viability as well as free calcium levels ([Ca2+]i) in short-term cultured hepatocytes. Reduced glutathione (GSH), adenosine-5′-triphosphate (ATP) and lipid peroxidation were investigated to evaluate the toxic effect of MDMA, in vitro, using freshly isolated rat hepatocytes. Methods: In order to measure cytosolic free Ca2+ concentrations ([Ca2+]i), rat hepatocytes were loaded with the Ca2+ indicator fura-2-acetoxymethylester (fura-2-AM). Results: A sustained rise of ([Ca2+]i) after incubation with MDMA was the most noteworthy finding. In Ca2+-free medium, MDMA caused a reduced increase of ([Ca2+]i). On the other hand, MDMA (0.1–5 mM) induced a concentration-dependent and time exposure-dependent GSH and ATP depletion. Although it did not reach statistical significance, GSH deficits were accompanied by a tendency to increase lipid peroxidation 3 h after MDMA incubation. Conclusions: The above data suggest that the marked rise of ([Ca2+]i) and subsequent ATP and GSH depletion can lead to a rapid decrease in cell viability.