In heart failure (HF) a main factor in reduced contractility is reduced SR Ca2+ content and reversed force-frequency response (FFR), ie, from positive to negative. Our arrhythmogenic rabbit HF model exhibits decreased contractility mainly due to an increase in Na/Ca exchange (NCX) activity (with only modest decrease in SR Ca2+-ATPase (SERCA) function), similar to many end-stage HF patients. Here we test whether phospholamban (PLB) inhibition using a dominant-negative mutant PLB adenovirus (K3E/R14E, AdPLB-dn, with β-galactosidase adenovirus as control) could enhance SERCA function and restore Ca2+ transients and positive FFR in ventricular myocytes from these HF rabbits. HF myocytes infected with AdPLB-dn (versus control) had enhanced Ca2+ transient amplitude (2.0±0.1 versus 1.6±0.05 F/Fo at 0.5 Hz, P<0.05) and had a positive FFR, whereas acutely isolated HF myocytes or those infected with Adβgal had negative FFR. Ca2+ transients declined faster in AdPLB-dn versus Adβgal myocytes (RT50%: 317±29 versus 551±90 ms at 0.5 Hz, P<0.05) and had an increased SR Ca2+ load (3.5±0.3 versus 2.6±0.2 F/Fo at 0.5 Hz, P<0.05), indicative of increased SERCA function. Furthermore, this restoration of function was not due to changes in NCX or SERCA expression. Thus, increasing SERCA activity in failing myocytes by AdPLB-dn gene transfer reversed the contractile dysfunction (and restored positive FFR) by increasing SR Ca2+ load. This approach could enhance contractile function in failing hearts of various etiologies, even here where reduced SERCA activity is not the main dysfunction. © 2005 American Heart Association, Inc.