Several studies have shown that peroxynitrite (ONOO– ), formed upon the reaction of •NO and O2–, is increased in many cardiovascular diseases and is detrimental to myocardial function. Proteins associated with Ca2+ homeostasis regulation in the heart may be involved in these effects. Thus, the aim of this study was to elucidate the mechanisms associated with ONOO– -induced effects. We evaluated [Ca2+]i regulation, sarco/endoplasmic reticulum Ca2+- binding proteins, and phosphorylation levels of the ryanodine receptor in isolated rat myocytes. Electrical field-induced intracellular Ca2+ transients and contractions were recorded simultaneously. Myocytes superfused with 3-morpholinosydnonimine N-ethylcarbamide (SIN-1), an ONOO– donor, decreased the amplitude of Ca2+ transients and contraction in a dose-response (1–200 µM) manner. Similarly, SIN-1 increased half-time decay in a concentration-dependent manner. Co-infusion of the ONOO– donor with FeTMPyP (1 µM), an ONOO– decomposition catalyst, inhibited the effects induced by ONOO– . Impaired sarcoplasmic reticulum Ca2+ uptake caused by ONOO– (SIN-1 200 µM) was confirmed by a reduction of caffeine-evoked Ca2+ release along with prolongation of the half-time decay. Surprisingly, ONOO– induced a spontaneous Ca2+ transient that started at the beginning of the relaxation phase and was inhibited by tetracaine. Also, reduced phosphorylation at the ryanodine receptor 2 (RyR2)-Ser-2814 site was observed. In conclusion, deficient sarco/endoplasmic reticulum Ca2+-ATPase-mediated Ca2+ uptake concomitant with augmented Ca2+ release by RyR2 in myocytes may be associated with modification of myocyte Ca2+ handling by ONOO– . Thus, development of cardiac failure in diabetes, nephropathy, or hypertension may be related with elevated ONOO–in cardiac tissue.