As a critical step toward understanding the role of abnormal intracellular Ca2+ release via the ryanodine receptor (RyR2) during the development of hypertension-induced cardiac hypertrophy and heart failure, this study examines two questions: 1) At what stage, if ever, in the development of hypertrophy and heart failure is RyR2 hyperphosphorylated at Ser2808? 2) Does the spatial distribution of RyR2 clusters change in failing hearts? Using a newly developed semiquantitative immunohistochemistry method and Western blotting, we measured phosphorylation of RyR2 at Ser2808 in the spontaneously hypertensive rat (SHR) at four distinct disease stages. A major finding is that hyperphosphorylation of RyR2 at Ser2808 occurred only at late-stage heart failure in SHR, but not in age-matched controls. Furthermore, the spacing between RyR2 clusters was shortened in failing hearts, as predicted by quantitative model simulation to increase spontaneous Ca2+ wave generation and arrhythmias.