Previously we have shown that the relationship between sarcoplasmic reticulum (SR) Ca content and Ca transient amplitude dissociates at high stimulation frequencies under current-clamp conditions (Dibb et al. 2003). This was not due to any change in action potential duration thus we have investigated if inactivation of the L-type Ca current or changes in intracellular [Na] were involved. The L-type Ca current was measured under voltage-clamp conditions using the perforated patch technique. Stimulation frequencies used encompassed the physiological range (4,6 and 8 Hz). Adult male rats were killed by cervical dislocation and ventricular myocytes were isolated by collagenase digestion. Simultaneous measurements of intracellular [Ca] and [Na] were made in myocytes co-loaded with Fluo-3 AM and SBFI AM respectively. SR Ca content was measured by rapid application of 10 mM caffeine at a holding potential of −80 mV (Varro et al. 1993). All experiments were performed at 37°C. Data are presented as mean ± SEM from n experiments. Statistical analysis was carried out using repeated measures ANOVA. Increasing the stimulation frequency over the range of 4 to 8 Hz resulted in a significant increase in diastolic Ca (p〈0.01) and a decrease in the amplitude of the systolic Ca transient (217±30, 153±23 & 87±17 nmol/l; p〈0.01 between all frequencies, n=10). Unlike our previous studies with current-clamp stimulation (Dibb et al. 2003) in this study there was no significant change in SR Ca content between 4, 6 and 8 Hz (92±8, 100±10 and 99±9 mmol/l; n=8-9). Thus, following stimulation from a holding potential of −40 mV changes in Ca transient amplitude were independent of SR Ca content. Intracellular [Na] increased with increasing stimulation frequency between 4, 6 and 8Hz (11.5▒1.2, 16.9▒1.4 and 21.0▒2.3 mM; p<0.05 between all frequencies, n=6-8) measured using field stimulation. Peak L-type Ca current decreased with increasing stimulation frequency from 4 to 8 Hz (5.2±0.7, 3.7±0.5 & 2.4±0.3 pA/pF; p〈0.05 between all frequencies, n=7). The rate of decay of the L-type current also decreased with increasing stimulation frequency from 4 to 8 Hz (9.5±0.6, 10.6±0.7 & 12.2±0.8; p〈0.05 between all frequencies, n=7). We propose that the reduced Ca transient amplitude therefore occurs as a consequence of the reduced trigger (L-type Ca current) and suggest that frequency dependent inactivation of the L-type Ca current may be responsible.