Intrinsic gender differences in cardiac function have been known to exist for some time, although the precise mechanisms responsible remain poorly understood. Recent evidence suggests that sex hormones can alter the expression and function of key proteins involved in intracellular Ca²⁺ regulation. We tested the hypothesis that gender differences in cardiac function can be related to differences in subcellular Ca²⁺ regulation, particularly at the level of the sarcoplasmic reticulum (SR) and Na⁺/Ca²⁺ exchanger (NCX). Electrically-induced and caffeine-induced Ca²⁺ transients were recorded in left ventricular myocytes isolated from humanely killed male and female rats and guinea-pigs using the Ca²⁺-sensitive fluorescent indicators indo-1 and fluo-4. Electrophysiological parameters were measured using discontinuous single electrode voltage-clamp. There was no significant gender difference in Ca²⁺ transient amplitude. Peak L-type Ca²⁺ current was smaller in females of the same age but the relative sizes of the current varied with maturation of the male. We measured SR Ca²⁺ contents by integrating the NCX current induced by rapid caffeine application. We found that SR Ca²⁺ content was greater in myocytes from females compared with those from males. This could be related to lower activity of the NCX in females, assessed by measuring repolarizing tail currents and Ca²⁺ transient decay kinetics following thapsigargin treatment. Further, females consistently display greater fractional release of Ca²⁺. The results allow us to begin to piece together the subcellular mechanisms responsible for gender differences in cardiac function.