Phospholamban has been suggested to be a key regulator of cardiac sarcoplasmic reticulum (SR) calcium cycling and contractility and a potential therapeutic target in restoring the depressed calcium cycling in failing hearts. Our understanding of the function of phospholamban stems primarily from studies in genetically altered mouse models. To evaluate the significance of this protein in larger mammalian species, which exhibit calcium cycling properties similar to human, we over-expressed phospholamban in adult rabbit cardiomyocytes. Adenoviral mediated gene transfer, at high multiplicities of infection, resulted in only 1.22-fold over-expression of phospholamban. There were no effects on twitch Ca-transient amplitude or decay under basal or isoproterenol stimulated conditions. Furthermore, the SR Ca-load and Na/Ca exchanger function were not altered. These apparent differences between phospholamban over-expression in rabbit, compared to previous findings in the mouse, may be due to a higher (1.5-fold) endogenous phospholamban/SERCA2a ratio and potential functional saturation of SERCA2a by phospholamban in rabbit cardiomyocytes. The findings suggest that important species-dependent differences in phospholamban regulation of SERCA2a occur. In larger mammals a higher fraction of SERCA2a pumps are regulated by phospholamban, and this may influence therapeutic strategies to enhance cardiac contractility and functional cardiac reserve.