Sarcoplasmic reticulum (SR) Ca 2+ cycling is tightly regulated by ryanodine receptor (RyR) Ca 2+ release and sarco-endoplasmic reticulum Ca 2+-ATPase (SERCA) Ca 2+ uptake during each excitation–contraction coupling cycle. We previously showed that RyR refractoriness plays a key role in the onset of SR Ca 2+ alternans in the intact rabbit heart, which contributes to arrhythmogenic action potential duration (APD) alternans. Recent studies have also implicated impaired SERCA function, a key feature of heart failure, in cardiac alternans and arrhythmias. However, the relationship between reduced SERCA function and SR Ca 2+ alternans is not well understood. Simultaneous optical mapping of transmembrane potential (V m) and SR Ca 2+ was performed in isolated rabbit hearts ( n = 10) using the voltage-sensitive dye RH237 and the low-affinity Ca 2+ indicator Fluo-5N-AM. Alternans was induced by rapid ventricular pacing. SERCA was inhibited with cyclopiazonic acid (CPA; 1–10 μM). SERCA inhibition (1, 5, and 10 μM of CPA) resulted in dose-dependent slowing of SR Ca 2+ reuptake, with the time constant ( tau) increasing from 70.8 ± 3.5 ms at baseline to 85.5 ± 6.6, 129.9 ± 20.7, and 271.3 ± 37.6 ms, respectively ( p < 0.05 vs. baseline for all doses). At fast pacing frequencies, CPA significantly increased the magnitude of SR Ca 2+ and APD alternans, most strongly at 10 μM (pacing cycle length = 220 ms: SR Ca 2+ alternans magnitude: 57.1 ± 4.7 vs. 13.4 ± 8.9 AU; APD alternans magnitude 3.8 ± 1.9 vs. 0.2 ± 0.19 AU; p < 0.05 10 μM of CPA vs. baseline for both). SERCA inhibition also promoted the emergence of spatially discordant alternans. Notably, at all CPA doses, alternation of SR Ca 2+ release occurred prior to alternation of diastolic SR Ca 2+ load as pacing frequency increased. Simultaneous optical mapping of SR Ca 2+ and V m in the intact rabbit heart revealed that SERCA inhibition exacerbates pacing-induced SR Ca 2+ and APD alternans magnitude, particularly at fast pacing frequencies. Importantly, SR Ca 2+ release alternans always occurred before the onset of SR Ca 2+ load alternans. These findings suggest that even in settings of diminished SERCA function, relative refractoriness of RyR Ca 2+ release governs the onset of intracellular Ca 2+ alternans.