In an attempt to evaluate the cardiotoxicity of bupivacaine, beating rate, tetrazolium MTT and lactate dehydrogenase activity were investigated in the medium containing bupivacaine for 24 hours after neonatal rat myocardial cells were cultured for 72 hours. Light and electron microscopic studies were also carried out. The results were as follows ; 1) Beating rate decreased dose-dependently, and beating cells were not observed over 10(-4) M concentration of bupivacaine. 2) MTT50 value was 0.32 ug/ml (1,000 uM). 3) The amount of lactate dehydrogenase released into the medium was 192% of control cells at 10(-3) M concentration of bupivacaine. 4. In light microscopy, myocardial cells were decreased in number dose-dependently, and showed a few cytoplasmic processes and lots of granules in cytoplasm at 10 M concentration of bupivacaine. 5. Electron microscopy of bupivacaine-treated cells showed smooth endoplasmic reticulum, destruction of mitochondria and Golgi apparatus and increase of vacuoles and dense bodies. It also showed dilatation of rough endoplasmic reticulum and loss of myofibrils. These results suggest that high concentration of bupivacaine (≥ 10(-4) M) induee remarkable toxicity on cultured rat myocardial cells.