Abstract Purkinje fibers play an essential role in transmitting electrical impulses through the heart, but they may also serve as triggers for arrhythmias linked to defective intracellular calcium (Ca 2+) regulation. Although prior studies have extensively characterized spontaneous Ca 2+ release in nondriven Purkinje cells, little attention has been paid to rate-dependent changes in Ca 2+ transients. Therefore we explored the behaviors of Ca 2+ transients at pacing rates ranging from 0.125 to 3 Hz in single canine Purkinje cells loaded with fluo3 and imaged with a confocal microscope. The experiments uncovered the following novel aspects of Ca 2+ regulation in Purkinje cells: 1) the cells exhibit a negative Ca 2+–frequency relationship (at 2.5 Hz, Ca 2+ transient amplitude was 66 ± 6% smaller than that at 0.125 Hz); 2) sarcoplasmic reticulum (SR) Ca 2+ release occurs as a propagating wave at very low rates but is localized near the cell membrane at higher rates; 3) SR Ca 2+ load declines modestly (10 ± 5%) with an increase in pacing rate from 0.125 Hz to 2.5 Hz; 4) Ca 2+ transients show considerable beat-to-beat variability, with greater variability occurring at higher pacing rates. Analysis of beat-to-beat variability suggests that it can be accounted for by stochastic triggering of local Ca 2+ release events. Consistent with this hypothesis, an increase in triggering probability caused a decrease in the relative variability. These results offer new insight into how Ca 2+ release is normally regulated in Purkinje cells and provide clues regarding how disruptions in this regulation may lead to deleterious consequences such as arrhythmias.