This study addressed the hypothesis that long-term deficiency of ovarian hormones alters cellular calcium (Ca2+) handling mechanisms in the heart resulting in the formation of a pro-arrhythmic substrate. It also tested if estrogen supplementation to ovariectomised guinea pigs reverses any alterations to cardiac Ca2+ handling and rescues pro-arrhythmic behaviour. Ovariectomy (OVx) or sham operations were performed on female guinea pigs using appropriate anaesthetic and analgesic regimes. Pellets containing 17β-estradiol (1mg, 60-day release), were placed subcutaneously in selected OVx animals (OVx+E). Cardiac myocytes were enzymatically isolated and electrophysiological measurements were conducted with a switch clamp system. In Fluo-4 loaded cells, Ca2+ transients were 20% larger and fractional sarcoplasmic reticulum (SR) Ca2+ release was 7% greater in the OVx group compared with the sham group. Peak L-type Ca2+ current (ICa,L) was 16% larger in OVx myocytes with channel inactivation shifting to more positive membrane potentials creating a larger "window" current. SR Ca2+ stores were 22% greater in the OVx group and these cells showed a higher frequency of Ca2+ sparks and waves and shorter wave-free intervals. OVx myocytes showed higher frequencies of early afterdepolarizations (EADs) and a greater percentage of these cells showed delayed afterdepolarizations (DADs) following exposure to isoprenaline (ISO) compared to sham. The altered Ca2+ regulation occurring in the OVx group was not observed in the OVx+E group. These findings suggest that long-term deprivation of ovarian hormones in guinea pig lead to changes in myocyte Ca2+ handling mechanisms that are considered pro-arrhythmogenic. 17β-estradiol replacement prevented these adverse effects.