Males and females show distinct differences in action potential waveform, ion channel expression patterns and ECG characteristics. However, it is not known how gender-based differences in Ca²⁺ cycling might contribute to these differences in electrophysiological activity. The goal of this study was to investigate the differences in cellular Ca²⁺ transients in males and females and to examine how these might contribute to electrophysiological function. Ca²⁺ transients were measured in individual myocytes within microscopic regions of the fluo-4AM loaded left ventricular epicardium of intact rat heart of both sexes (3-4 months old). Pacing protocols were used to measure transient characteristics at a basic cycle length of 500msec and during 10-second trains of rapid pacing delivered to the LV apex. Ca²⁺ transients were smaller in magnitude and longer in duration in females than in males. More importantly, variability in Ca²⁺ transient characteristics between myocytes in a microscopic recording site (heterogeneity index) was greater for females than males for characteristics related to transient duration. The rate-sensitivity of Ca²⁺ alternans development in individual myocytes was greater in females than in males but there was also greater heterogeneity in cellular responses to the rate-dependence of alternans development in females. The longer Ca²⁺ transients in females were also associated with slower restitution which was likely to be responsible for the development of Ca²⁺ and repolarization alternans at slower heart rates. These results demonstrate that there are distinct differences in cellular Ca²⁺ cycling in male and female rat hearts. Not only is there slower reuptake of Ca²⁺ in female rats but there is greater local variability in Ca²⁺ cycling at the microscopic level. These gender-based differences in Ca²⁺ cycling could contribute to differences in ECG morphology and in arrhythmia sensitivity in males and females.