Inadvertent intravenous administration of bupivacaine, unlike that of lidocaine, is associated with significant cardiotoxicity. However, the mechanism(s) underlying this phenomenon is uncertain. High concentrations of cardiolipin, an anionic phospholipid, are found in the mitochondria membrane of cardiomyocytes. We hypothesized that bupivacaine, but not lidocaine, interacts avidly with cardiolipin in the mitochondria membrane of cardiomyocytes and alters its integrity thereby accounting, in part, for cardiotoxicity. Accordingly, the purpose of this study was to begin to address this issue by determining the effects of bupivacaine and lidocaine on permeability of cardiolipin-containing biomimetic small unilamellar liposomes. We found that bupivacaine, but not lidocaine, elicited a significant, concentration-dependent increase in carboxyfluorescein release from cardiolipin-containing small unilamellar liposomes (size, 165nm) composed of egg yolk phosphatidylcholine and cholesterol (p<0.05). Both drugs had no significant effects on carboxyfluorescein release from liposomes devoid of cardiolipin (p>0.5). Collectively, these data indicate that bupivacaine, but not lidocaine, interacts avidly and selectively with biomimetic small unilamellar liposomes containing cardiolipin and disrupts their integrity. We suggest that these interactions underlie, in part, bupivacaine-induced cardiotoxicity.