Action potential duration (APD) restitution properties and repolarization alternans are thought to be important arrhythmogenic factors. We investigated the role of intracellular calcium (Ca²⁺_i) cycling in regulating APD restitution slope and repolarization (APD) alternans in patch-clamped rabbit ventricular myocytes at 34 to 36°C, using the perforated or ruptured patch clamp techniques with Fura-2-AM to record Ca²⁺_i. When APD restitution was measured by either the standard extrastimulus (S1S2) method or the dynamic rapid pacing method, the maximum APD restitution slope exceeded 1 by both methods, but was more shallow with the dynamic method. These differences were associated with greater Ca²⁺_i accumulation during dynamic pacing. The onset of APD alternans occurred at diastolic intervals at which the APD restitution slope was significantly <1 and was abolished by suppressing sarcoplasmic reticulum (SR) Ca²⁺_i cycling with thapsigargin and ryanodine, or buffering the global Ca²⁺_i transient with BAPTA-AM or BAPTA. Thapsigargin and ryanodine flattened APD restitution slope to <1 when measured by the dynamic method, but not by the S1S2 method. BAPTA-AM or BAPTA failed to flatten APD restitution slope to <1 by either method. In conclusion, APD alternans requires intact Ca²⁺_i cycling and is not reliably predicted by APD restitution slope when Ca²⁺_i cycling is suppressed. Ca²⁺_i cycling may contribute to differences between APD restitution curves measured by S1S2 versus dynamic pacing protocols by inducing short-term memory effects related to pacing-dependent Ca²⁺_i accumulation.