The type-I ribosome-inactivating protein trichosanthin (TCS) has a broad spectrum of biological and pharmacological activities, including abortifacient, anti-tumour and anti-HIV activities. We have found for the first time that TCS stimulated the production of reactive oxygen species (ROS) in JAR cells (a human choriocarcinoma cell line) in a time- and concentrationdependent manner by using the fluorescent probe 2',7'-dichlorofluorescein diacetate with confocal laser scanning microscopy. ESR spectral studies and the inhibition of ROS formation by the superoxide radical anion (O2 −d) scavenger superoxide dismutase, the H3O2 scavenger catalase and the hydroxyl radical (OHd) scavenger mannitol suggested the involvement of O2−d, H2O2 and OHd. TCS-induced ROS formation was shown to be dependent on the presence of both extracellular and intracellular Ca2+; moreover, ROS production paralleled the intracellular Ca2+ elevation induced by TCS, suggesting that ROS production might be a consequence of Ca2+ signalling. TCS-induced activation of caspase-3 was initiated within 2 h; however, TCS-induced production of ROS was initiated within 5min, suggesting that the production of ROS preceded the activation of caspase-3. Simultaneous observation of the nuclear morphological changes via two-photon laser scanning microscopy and ROS production via confocal laser scanning microscopy revealed that ROS is involved in the apoptosis of JAR cells. The involvement of ROS was also confirmed by the inhibition of TCS-induced cell death by the antioxidant Trolox and the ROS scavengers catalase and mannitol. Diethylenetriaminepenta-acetic acid, an inhibitor of metal-facilitated OHd formation, markedly inhibited TCS-induced cell death, suggesting that TCS induced OHd formation via the Fenton reaction. The finding that ROS is involved in the TCS-induced apoptosis of JAR cells might provide new insight into the antitumour and anti-HIV mechanism of TCS.