Possible apoptosis (programmed cell death) caused by MDMA in liver cells was investigated in cultured rat hepatocytes and hepatic stellate cells (HSC). Hepatocytes came from Wistar rats, and HSC came from an established cell line. Cell cultures were treated with 0.5, 1, 3, or 5 mM MDMA, and incubation lasted either for 8 or 24 hours. Potential apoptotic activity was assayed through multiple measures. Methods included cell nucleus staining (to spot DNA fragmentation), assessing soluble histone-DNA complex, Western blot (immunoassay using chemiluminescence autoradiography) for bcl-x (inhibitors of pro-apoptotic factors) and bax (promoters of apoptotic activity), and assessing caspase 3 activity. Hepatocytes showed morphological changes after 1 mM MDMA at 8 h incubation, as determined by visual inspection, but HSC only began showing signs of morphological changes at 5 mM MDMA at 24 h incubation. All treatments produced some signs of chromatin condensation and nuclear fragmentation, though hepatocytes were more sensitive than HSC. Applying a caspase-3 inhibitor (Ac-DEVD-CHO) reduced signs of nuclear fragmentation. MDMA treatment reduced expression of the apoptosis inhibitor bcl-x, but did not increase expression of the pro-apoptotic factor bax in hepatocytes incubated for 8 h with 0.5 or 1 mM MDMA or in HSC incubated for 24 h with 3 or 5 mM MDMA. MDMA treatment (at same concentrations used in the bcl and bax assays) also enhanced cytochrome C and increased caspase 3 activity. While signs of the execution phase of apoptosis were seen in HSC cultures incubated with 3 or 5 nM MDMA for 8 h, it was not seen in hepatocytes incubated with 0.5 or 1 mM MDMA for 8 h. Study findings indicate a role for apoptotic processes, perhaps induced by oxidative stress, in MDMA-induced hepatotoxicity. However, the authors acknowledge that the concentrations used in their studies are higher than levels producing neurotoxicity in rats (15 mcM). The concentrations used in this report were at least 11 times greater than concentrations found in humans. Hence it seems unlikely that apoptotic action alone is a plausible explanation for ecstasy-related hepatotoxicity in humans, given its relative rarity and association with hyperthermia.