Na⁺/Ca²⁺ exchanger (NCX) protein level and activity were measured in myocardium from the basal region of the left ventricle of rabbit hearts with significant left ventricular dysfunction (LVD) 8-9 weeks after an apical infarction. NCX protein abundance was higher in the tissue homogenates (121±11%) and purified membrane fractions (143±12%) in LVD compared to the sham group. NCX mRNA was also higher in the LVD group (126%). Lower NCX protein expression was observed in the membrane fractions from the epicardium compared to the endocardium in both sham and LVD groups. Trans-membrane currents were recorded in isolated cardiomyocytes by single-electrode voltage-clamp; [Ca²⁺]_i was measured using Fura-2. Rapid application of 10mmol/L caffeine was used to induce SR Ca²⁺ release. The subsequent NCX- mediated Ca²⁺ efflux rate constant was lower (70% of sham) in the LVD group. NCX currents were measured in cardiomyocytes dialysed with 250nM Ca²⁺ (50mmol/L EGTA). A lower NCX current (75% of sham) was observed in the LVD group. Lower NCX activity was also observed in cardiomyocytes isolated from the epicardium compared to the endocardium; a transmural difference that was also seen in the LVD group. Reduced activity despite increased protein expression may result from reduced Ca²⁺ sensitivity of the allosteric regulation of NCX. However, measurements indicated increased Ca²⁺ sensitivity in the LVD group. Cardiomyocytes from LVD hearts displayed a marked reduction in transverse tubule area (59% of sham), and surface-area/volume ratio (80% of sham). Disrupted transverse tubule structure may contribute to the decrease in NCX activity despite increased protein expression in LVD.