Objectives: To assess the phosphorylation of myocardial phospholamban (PLB) and quantify protein levels of PLB and sarco/endoplasmic reticulum Ca²⁺ ATPase type 2 (SERCA 2) in a rabbit model of heart failure. Furthermore, to correlate these parameters with the rate of Ca²⁺ uptake into sarcoplasmic reticulum (SR) vesicles. Methods: Heart failure in the rabbit was indicated by the pronounced ventricular contractile dysfunction accompanied by post-mortem evidence of lung and liver congestion 8 weeks after a coronary artery ligation procedure. Phosphorylation of PLB was measured by reduced mobility of the phosphorylated forms on Tris–glycine gels. Phosphoserine and phosphothreonine-specific antibodies against PLB were used to determine the phosphorylated residues. Immunoblotting combined with densitometry was used to assess PLB and SERCA 2 levels. Finally, oxalate-supported Ca²⁺ uptake into SR vesicles was studied using the fluorescent indicator Fura-2. Results: The phosphorylation state of PLB was significantly higher in myocardium isolated from left ventricles of heart failure rabbits (8.3±0.42 P-PLB) when compared with sham-operated animals (4.0±1.7 P-PLB). The kinase activity associated with SR vesicles isolated from animals with heart failure was a factor of 1.58±0.21-times higher than sham hearts, as assessed by the initial rate of phosphorylation of PLB. This higher kinase activity observed in heart failure was not completely abolished by inhibitors of either A-kinase, C-kinase or Ca²⁺/calmodulin-dependent protein kinase (CaM-kinase). Abundance of SERCA in heart failure myocardial homogenates was significantly less than sham values (0.68±0.11 vs. 1.74±0.27) as was PLB (0.41±0.08 vs. 0.69±0.13), similar reductions were seen in vesicle preparations. The rate constant of Ca²⁺ uptake into the isolated SR vesicles was lower in preparations from heart failure myocardium than from sham myocardium (2.50±0.23 ms vs. 4.43±0.3 ms). Conclusions: The higher level of phosphorylation of PLB observed in the left ventricle of rabbits with heart failure is associated with a higher intrinsic kinase activity of the SR. However, the abundance of both of SERCA 2 and PLB proteins are lower in heart failure. The net effect of these changes appears to be a reduced rate of Ca²⁺ uptake by the SR in heart failure.