Sorcin is a 22 KDa EF hand Ca²⁺-binding protein expressed in cardiac tissue that modulates excitation-contraction (EC) coupling in cardiomyocytes by interacting with RyR2, NCX and I_CaL. I_CaL is a critical Ca²⁺ influx pathway which dictates the Ca²⁺ load of the cell. One way of controlling Ca²⁺ influx is by regulating current inactivation. Slowing of current inactivation and increases in peak current underlie I_CaL facilitation which occurs during increases in stimulation rate. This effect contributes to the positive contraction-frequency relationship and is thought to be the result of CaMKII-mediated phosphorylation of I_CaL. Sorcin has also been shown to modulate I_CaL inactivation kinetics and so this study was performed to investigate the effects of CaMKII and sorcin on I_CaL. Rabbit ventricular myocytes were whole-cell patch clamped using Cs- and TEA-rich solutions with 50 mM EGTA (170 nM free Ca²⁺) in the patch pipette to spatially restrict diffusion of Ca²⁺ to ~40nm and prevent overlap of K⁺ and Na⁺ currents with I_CaL, which was activated by a depolarising voltage step (-80 mV to 0 mV). Myocytes displayed I_CaL facilitation following an increase in stimulation frequency from 0.1 Hz to 1 Hz: the peak current ratio of beat 5 and beat 1 (B5/B1) was 1.2±0.02 (n=8) and inactivation kinetics were slowed from 6±0.5 ms (beat 1) (n=8) to 13.3±2 ms (beat 5) (n=8, P<0.05). 5 μM KN-93 (a CaMKII inhibitor) abolished current facilitation: B5/B1 was 0.88±0.02 (n=8, P0.05). This data confirms the involvement of CaMKII in mediating facilitation of I_CaL. Sorcin (3 μM) dialysed into the cell via the patch pipette, prolonged I_CaL inactivation and did not prevent current facilitation (Control beat 1, 6±0.5 ms, n=8; Sorcin beat 1, 12.7±2.3 ms, n=6, P<0.05; Control beat 5, 13.3±2 ms, n=8; Sorcin beat 5, 21.5±2 ms, n=6, P<0.05). We investigated the involvement of CaMKII in the actions of sorcin by using KN-93. B5/B1 was significantly lower in the presence of KN-93 (Sorcin, 1.18±0.05, n=6; Sorcin + KN-93, 0.92±0.02, n=7, P<0.05), suggesting that facilitation was inhibited. However, current inactivation was still prolonged at beat 5 compared to beat 1 (Sorcin + KN-93 beat 1, 8.8±0.4 ms; Sorcin + KN-93 beat 5, 18.8±2.2 ms, n=7, P<0.05), suggesting that despite inhibition by KN-93, sorcin can still influence current inactivation kinetics. This data suggests that sorcin does not modulate ICaL kinetics via the CaMKII pathway and that I_CaL facilitation operates within a cellular microdomain.