Objective: In heart failure (HF), the generation of reactive oxygen species (ROS) is enhanced. It was shown that failing cardiac myocytes are more susceptible to ROS-induced damage, possibly due to increased expression of the sarcolemmal Na-Ca exchanger (NCX). Methods: We investigated the consequences of increased expression levels of NCX in adult rabbit ventricular cardiomyocytes (via adenovirus-mediated gene transfer, Ad-NCX1-GFP) with respect to tolerance towards ROS. After 48-h incubation, cells were monitored for morphological changes on an inverted microscope. ROS were generated via hydrogen peroxide (H 2 O 2 ) (100 μmol/l) and Fe 3 + /nitrilotriacetate (Fe 3 + /NTA, 100/200 μmol/l) for 4 min and cell morphology was followed over 30 min. [Na + ] i and [Ca 2 + ] i in native cells were measured using SBFI-AM and Indo1-AM, respectively. Results: In native myocytes, exposure to ROS induced hypercontracture. This was accompanied by a 1.3-fold increase in diastolic Indo1 fluorescence ratio (P<0.05). Overexpression of NCX significantly enhanced development of hypercontracture. After 15 min, the percentage of cells that had undergone hypercontracture (F h y p e r ) was 85+/-4% vs. only 44+/-10% in control cells (P<0.05). Inhibition of NCX-mediated Ca 2 + entry with KB-R7943 (5 μmol/l) reduced F h y p e r to 33+/-11% (P<0.05). [Na + ] i was increased 2.9-fold 1 min prior to hypercontracture (P<0.05). Conclusions: ROS-induced hypercontracture is due to Ca 2 + entry via NCX which could be triggered by a concomitant substantial increase in [Na + ] i . Elevated NCX levels predispose to ROS-induced injury, a mechanism likely contributing to myocyte dysfunction and death in heart failure.