Objective: Phospholamban ablation or ectopic expression of SERCA1a in the heart results in significant increases in cardiac contractile parameters. The aim of the present study was to determine whether a combination of these two genetic manipulations may lead to further augmentation of cardiac function. Methods: Transgenic mice with cardiac specific overexpression of SERCA1a were mated with phospholamban deficient mice to generate a model with SERCA1a overexpression in the phospholamban null background (SERCA1_OE/PLB_KO). The cardiac phenotype was characterized using quantitative immunoblotting, sarcoplasmic reticulum calcium uptake and single myocyte mechanics and calcium kinetics. Results: Quantitative immunoblotting revealed an increase of 1.8-fold in total SERCA level, while SERCA2 was decreased to 50% of wild types. Isolated myocytes indicated increases in the maximal rates of contraction by 195 and 125%, the maximal rates of relaxation by 200 and 124%, while the time for 80% decay of the Ca²⁺-transient was decreased to 43 and 75%, in SERCA1_OE/PLB_KO hearts, compared to SERCA1a overexpressors and phospholamban knockouts, respectively. These mechanical alterations reflected parallel alterations in V_max and EC₅₀ for Ca²⁺ of the sarcoplasmic reticulum Ca²⁺ transport system. Furthermore, there were no significant cardiac histological or pathological alterations, and the myocyte contractile parameters remained enhanced, up to 12 months of age. Conclusions: These findings suggest that a combination of SERCA1a overexpression and phospholamban ablation results in further enhancement of myocyte contractility over each individual alteration.