Abstract: In this study, we applied acute toxicity test to investigate the toxic effects of copper ion(Cu2+) on tricarbo-xylic acid cycle(TCA) and free radical metabolism in the mitochondria of the liver of Hoplobatrachus chinensis. We treated the animals with Cu2+ at different concentrations and for different exposure time, and tested the activities of TCA-related enzymes and free radicals. We found that although Cu2+ at high concentrations could reduce the activity of ICDHm, prolonged exposure time had no significant effect on ICDHm(P 0.05). We did not observe cross effects between the concentration of Cu2+ and the exposure time on the activity of ICDHm(P 0.05). However there were significant cross effects between the exposure time and the concentration of Cu2+ on the activity of -KGDH(P 0.05). The activity of -KGDH decreased when exposed to Cu2+, and the lowest activities were detected when the exposure times were 24h and 96h and concentrations were 4.0 and 6.0 mg/L. In terms of free radical metabolism, the exposure time and copper concentration had strong cross effects on the activities of anti-O2 and H2O2 (P 0.05), but not on the activity of inhibit-OH, the content of NO, and the activity of NOS. The activities of anti-O2 decreased along with the increase in the concentration of Cu2+. The content of H2O2 increased after the treatments and perked at 24h and 6.0 mg/L. There was a negative correlation between the activity of inhibit-OH and the exposure time and the concen-tration of Cu2+. Along with the increase in the concentration of Cu2+, the content of NO and the activity of NOS first increased and then decreased to the values of the control group, and the maximum values appeared in the 6.0 mg/L group. Our study demonstrated that the acute exposure of Cu2+ could have significant toxic effects on TCA cycle and free radical metabolism in the mitochondria of the liver of H. chinensis.