Ca(2+) released from the sarcoplasmic reticulum (SR) via ryanodine receptor type 2 (RYR2) is the key determinant of cardiac contractility. Although activity of RYR2 channels is primary controlled by Ca(2+) entry through the plasma membrane, there is growing evidence that Ca(2+) in the lumen of the SR can also be effectively involved in the regulation of RYR2 channel function. In the present study, we investigated the effect of luminal Ca(2+) on the response of RYR2 channels reconstituted into a planar lipid membrane to caffeine and Ca(2+) added to the cytosolic side of the channel. We performed two sets of experiments when the channel was exposed to either luminal Ba(2+) or Ca(2+). The given ion served also as a charge carrier. Luminal Ca(2+) effectively shifted the EC(50) for caffeine sensitivity to a lower concentration but did not modify the response of RYR2 channels to cytosolic Ca(2+). Importantly, luminal Ca(2+) exerted an effect on channel gating kinetics. Both the open and closed dwell times were considerably prolonged over the whole range (response to caffeine) or the partial range (response to cytosolic Ca(2+)) of open probability. Our results provide strong evidence that an alteration of the gating kinetics is the result of the interaction of luminal Ca(2+) with the luminally located Ca(2+) regulatory sites on the RYR2 channel complex.