Sarcolemmal CD36 facilitates myocardial fatty acid (FA) uptake, which is markedly reduced in CD36-deficient rodents and humans. CD36 also mediates signal transduction events involving a number of cellular pathways. In taste cells and macrophages, CD36 signaling was recently shown to regulate store-responsive Ca2+ flux and activation of Ca2+-dependent phospholipases A2 that cycle polyunsaturated FA into phospholipids. It is unknown whether CD36 deficiency influences myocardial Ca2+ handling and phospholipid metabolism, which could compromise the heart, typically during stresses. Myocardial function was examined in fed or fasted (18-22 h) CD36 -/- and WT mice. Echocardiography and telemetry identified conduction anomalies that were associated with the incidence of sudden death in fasted CD36-/- mice. No anomalies or death occurred in WT mice during fasting. Optical imaging of perfused hearts from fasted CD36-/- mice documented prolongation of Ca2+ transients. Consistent with this, knockdown of CD36 in cardiomyocytes delayed clearance of cytosolic Ca 2+. Hearts of CD36-/- mice (fed or fasted) had 3-fold higher SERCA2a and 40% lower phospholamban levels. Phospholamban phosphorylation by protein kinase A (PKA) was enhanced after fasting reflecting increased PKA activity and cAMP levels in CD36-/- hearts. Abnormal Ca2+ homeostasis in the CD36-/- myocardium associated with increased lysophospholipid content and a higher proportion of 22:6 FA in phospholipids suggests altered phospholipase A2 activity and changes in membrane dynamics. The data support the role of CD36 in coordinating Ca2+ homeostasis and lipid metabolism and the importance of this role during myocardial adaptation to fasting. Potential relevance of the findings to CD36-deficient humans would need to be determined.