The sodium / calcium exchanger (NCX) is discussed as one of the key proteins involved in heart failure. However, the causal role and the extent to which NCX contributes to contractile dysfunction during heart failure are poorly understood. NCX overexpression was induced by infection with an adenovirus coding for NCX, which co-expressed GFP (AdNCX), by ex vivo gene transfer to non-failing and failing rabbit cardiomyocytes. Myocardial gene transfer in rabbits in vivo was achieved by adenoviral delivery via aortic cross-clamping. Peak cell shortening (pCS) of cardiomyocytes was determined photo-optically. Haemodynamic parameters in vivo were determined by echocardiography (fractional shortening, FS) and tip catheter (maximal first derivative of LV pressure; dp/dt max; maximal negative derivative of LV pressure; -dp/dt max). pCS was depressed after NCX gene delivery in isolated non-failing and in failing cardiomyocytes. In non-failing rabbits in vivo, basal systolic contractility (FS and dp/dt max) and maximum rate of LV relaxation (-dp/dt max) in vivo was largely unaffected after NCX overexpression. However, during heart failure, NCX overexpression over 2 weeks significantly improved FS, dp/dt max and -dp/dt max compared to AdGFP-infected failing rabbits. These results indicate that short-term effects of NCX overexpression impair contractility of isolated failing and non-failing rabbit cardiomyocytes. NCX overexpression over 2 weeks in vivo does not seem to affect myocardial contractility in non-failing rabbits. Interestingly, in vivo overexpression of NCX decreased the progression of systolic and diastolic contractile dysfunction, and contractile reserve in heart failure in rabbits in vivo.