Microcystin-LR (MCLR), the most toxic microcystin up to date, could induce apoptosis in many kinds of fish and mammalian cells. For the fish immunotoxicity, it was found that MCLR could induce apoptosis in Carassius auratus lymphocytes in vitro. So this study focused on the role of intracellular Ca(2+), mitochondrial membrane potential, reactive oxygen species (ROS), and intracellular ATP in response to the mechanisms of MCLR-induced apoptosis in fish lymphocytes. MCLR (10 nM) administration resulted in a massive elevation in ROS, intracellular Ca(2+), decreased ATP, and rapid mitochondrial membrane potential (DeltaPsi(m)) disruption. When compared to controls, both a fourfold significant (P < 0.001) elevation in O(2) (-) in 1.5 h and an approximately twofold increase in Ca(2+) in 0.5 h were observed. After 6 h of treatment, an approximately 30% decrease for DeltaPsi(m) but about 75% decline for ATP were found. Together, the results demonstrated that MCLR-induced apoptosis was associated with a massive calcium influx, resulting in O(2) (-) elevation, DeltaPsi(m) disruption, and ATP depletion. This study provided a possible cytotoxic mechanism of fish lymphocytes caused by MCLR.