Phospholamban forms an integral part of the cardiac sarcoplasmic reticulum (SR) and regulates the activity of SR Ca²⁺-ATPase (SERCA2a). A number of studies have suggested a decrease in SERCA2a relative to phospholamban in heart failure. To test the hypothesis that changes in the relative abundance of phospholamban to SERCA2a could account for the pathophysiological abnormalities in Ca²⁺ handling observed in failing myocardium, we created a recombinant adenovirus designed to overexpress phospholamban (Ad.RSV.PL). In neonatal rat cardiomyocytes, Ad.RSV.PL increased the expression of phospholamban in a concentration-dependent fashion, reaching 280±43% at a multiplicity of infection (MOI) of 10.0 plaque forming units (pfu)/cell at 48 hours. The relationship between Ca²⁺-ATPase activity and [Ca²⁺] was shifted rightward in membrane preparations from cardiomyocytes infected with Ad.RSV.PL. Intracellular Ca²⁺ transients measured in the neonatal cells infected with Ad.RSV.PL (MOI, 10 pfu/cell) were characterized by (1) a significant prolongation of the relaxation phase (344±26 versus 710±56 milliseconds, P<.01), (2) a decrease in peak [Ca²⁺]_i (967±43 versus 630±33 nmol/L, P<.01), and (3) an elevation in resting [Ca²⁺]_i (143±14 versus 213±17 nmol/L, P<.05). Similarly, the time course of shortening was prolonged in myocytes infected with Ad.RSV.PL. These effects were partially restored by simultaneous transduction with an adeno-virus carrying SERCA2a. Cardiomyocytes infected with Ad.RSV.PL had an abnormal frequency response: a decrease in peak [Ca²⁺]_i and an increase in resting [Ca²⁺]_i with increasing frequency. These findings indicate that adenovirus-mediated gene transfer of phospholamban modifies intracellular Ca²⁺ handling and the frequency response in cardiomyocytes. Our results suggest that alterations in the ratio of phospholamban to SERCA2a could account for the abnormalities in Ca²⁺ handling observed in heart failure and that overexpression of SERCA2a can largely correct these abnormalities.