Mammalian hearts show important age-related deficits in contractile function, in particular when heart rates are rapid (Lakatta, 2003). Previous studies have shown that these defects in contractile function also are present in ventricular myocytes isolated from ageing hearts (Xiao et al., 1994; Lim et al., 2000; Isenberg et al., 2003; Dibb et al., 2004). However, previous studies of excitation-contraction (EC)-coupling in ageing myocytes have been conducted in cells isolated from male animals only, female animals only or from animals where the sex has not been specified. Thus, whether ageing affects cardiac EC-coupling differently in myocytes from male and female animals has not yet been investigated. The goals of this study were to characterize age-related alterations in EC-coupling in ventricular myocytes and to investigate whether these alterations were affected by the sex of the animal. Experiments utilized myocytes isolated from young adult (7 months) and aged (24 months) male and female B6SJLF1/J mice or young adult and aged male Fischer 344 rats. All studies were performed at 37 degrees Celsius. Voltage clamp experiments were conducted with high resistance microelectrodes in isolated ventricular myocytes. Cells were paced with a series of conditioning pulses delivered at a frequency of 2 Hz prior to test steps from -40 to 0 mV. Myocytes were loaded with fura-2 AM to measure intracellular calcium concentrations. Sarcoplasmic reticulum (SR) calcium content was assessed by rapid application of 10 mM caffeine. Unloaded cell shortening was measured with a video edge detector. Results showed that the amplitudes of fractional cell shortening and calcium current densities were significantly smaller in aged male myocytes when compared to cells from younger males (mean calcium current densities were -6.0 ± 0.4 vs. -3.2 ± 0.9 pA/pF for young adult and aged male mice; p<0.05; n=13/group; values were -8.2 ± 0.7 vs. -6.2 ± 0.4 pA/pF for young adult and aged male rats; p<0.05; n=16-23/group). In addition, calcium transients were significantly smaller and had slower rise times in aged male myocytes than in younger adult cells (mean calcium transient amplitudes were 167.4 ± 22.4 vs. 83.0 ± 23.2 nM for young adult and aged male mice; p<0.05; n=7-10/group). Similar results were obtained in myocytes from young adult and aged rats. Systolic and diastolic calcium concentrations also were significantly lower in aged male myocytes than in cells from younger animals. Interestingly, SR calcium content declined with age in male myocytes. However, the amount of calcium released per unit calcium current (an estimate of EC-coupling gain) was similar in young adult and aged male cells (values were 828.0 ± 180.9 vs. 715.2 ± 237.3 nM sec^-1/pA/pF^-1 for young adult and aged male mice; p<0.05; n=7-9/group). EC-coupling gain also was decreased significantly in aged male rat cells when compared to younger myocytes (values were 314.4 ± 62.9 vs. 178.8 ± 25.3 nM sec^-1/pA/pF^-1 for young adult and aged male rats; p<0.05; n=16-23/group). Furthermore, the fractional release of SR calcium was similar in young adult and aged male cells. These results show that there is an age-related decrease in cardiac contractile function in ventricular myocytes from male mice and rats. Results in myocytes obtained from female animals were markedly different. In contrast to results in male animals, fractional shortening and calcium current densities were similar in young adult and aged myocytes isolated from female hearts (mean calcium current densities were -4.6 ± 0.4 vs. -3.5 ± 0.5 pA/pF for young adult and aged female mice; n=11-20/group). Furthermore, calcium transient amplitudes and rates of rise were unaffected by age in female cells (mean calcium transient amplitudes were 140.7 ± 23.5 vs. 112.0 ± 16.2 nM for young adult and aged female mice; n=9-21/group). In addition, systolic and diastolic calcium concentrations were similar in young adult and aged female myocytes. Interestingly, SR calcium content actually was elevated in aged female myocytes compared to cells from younger animals and fractional SR calcium release declined with age in females. However, the gain of EC coupling was similar in myocytes from young adult and aged female mice (values were 859.6 ± 272.3 vs. 909.5 ± 145.4 nM sec^-1/pA/pF^-1 for young adult and aged females; n=9-21/group). These data demonstrate that age-related deficits in cardiac EC-coupling are prominent in ventricular myocytes from male hearts but not in cells from female hearts. Thus, age-related changes in cardiac EC-coupling in murine ventricular myocytes are influenced markedly by the sex of the animal. These observations suggest that it is important to consider sex as a variable in studies of the effects of ageing on cardiac EC-coupling.