The heart spends about half of its time in diastole as relaxation is required for normal cardiac function. Impairment of relaxation can lead to diastolic heart failure. Although much is known about the regulation of systolic Ca concentration ([Ca2+]i), less is known about the regulation of diastolic. In a recent study on rat ventricular myocytes, we found that the time-averaged level of [Ca2+]i was determined by Ca influx into the cell, thereby controlling diastolic [Ca2+]i [Sankaranarayanan et al.]. In this study, therefore, we aimed to investigate the effects of a simple inotropic manoeuvre, elevation of external Ca concentration, on both diastolic and systolic calcium concentration. Cardiac ventricular myocytes were isolated from mice and loaded with the AM form of the fluorescent calcium indicator Fluo-3. Calcium transients and membrane currents were recorded with the perforated patch clamp technique. Different stimulation frequencies (0.5, 3 Hz) were achieved by stimulating the cells through the patch electrode with square depolarizing pulses. External Ca concentration was varied over the range 0.5 to 5 mM. Data are reported as the mean ± SEM for “n” cells. At normal (1 mM) or low (0.5 mM) external Ca, increasing frequency from 0.5 to 3 Hz decreased the amplitude of the Ca transient while increasing diastolic [Ca2+]i. (For example, at 0.5 mM Ca, increasing frequency decreased amplitude from 322±60 nM to 133±22 nM; n=16, P<0.001 and increased diastolic from 76±12 nM to 84±10 nM; n=15, P<0.005). In contrast, at 5 mM Ca, increasing frequency had no effect on the amplitude (567±88 nM vs 571±75 nM; n=10, NS) but still increased diastolic [Ca2+]i (from 203±46 nM to 407±95 nM; n=9, P<0.05). At all Ca concentrations, however, increasing frequency increased the time-averaged [Ca2+]i as has been found in rat myocytes. These changes of time-averaged [Ca2+]i were compared with the measured Ca influx per unit time, estimated from the L-type Ca current. Increasing frequency increased Ca influx per unit time. The fractional change of measured influx was, however, greater than that of average [Ca2+]i suggesting a contribution of a significant, frequency-independent Ca influx mechanism in addition to the L-type Ca current [Sankaranarayanan et al.]. These results demonstrate that changes of time-averaged [Ca2+]i result from those of Ca influx. Future work needs to identify this additional Ca influx pathway and how it is affected by physiological and pathological changes.