To compare the cardiotoxicity induced by ropivacaine and bupivacaine and to investigate the mechanism of cardiotoxicity, 24 mature New Zealand rabbits were divided randomly into control group (group C), ropivacaine group (group R) and bupivacaine group (group B). Hearts were drawn out rapidly from the anesthetized animals and cardiac perfusion was performed immediately. Ropivacaine 500 ng/ml (group R) or bupivacaine 500 ng/ml (group B) was added to the perfusion solution. Ventricular myocardial ATP, ADP and AMP were measured with high performance liquid chromatogram. The ability of myocardial mitochondria oxidation to pyruvate or palmitoylcarnitine was detected with Clark electrode. Our results showed that myocardial ATP and ADP decreased significantly (P < 0.05) in group R and most significantly (P < 0.01) in group B as compared with group C. Myocardial ATP and ADP decreased most significantly (P < 0.01) in group B as compared with group R. The changes of myocardial AMP revealed significant difference among three groups. The changes of pyruvate oxidation exhibited no significant difference among the three groups. Palmitoylcarnitine oxidation decreased markedly (P < 0.05) in group R and most significantly (P < 0.01) in group B as compared with group C. The present study indicated that the inhibition of lipid substrate oxidation may be responsible for the cardiotoxicity induced by bupivacaine and ropivacaine. The cardiotoxicity induced by ropivacaine is far more less than bupivacaine.