We investigated the possibility that the Ca²⁺ channel agonist FPL-64176 (FPL) might also activate the cardiac sarcoplasmic reticulum (SR) Ca²⁺ release channel ryanodine receptor (RyR). The effects of FPL were tested on single channel activity of purified and crude vesicular RyR (RyR2) isolated from human and dog hearts using the planar lipid bilayer technique. FPL (100-200 µM) increased single channel open probability (P_o) when added to the cytoplasmic side of the channel (P_o = 0.070 ± 0.021 in control RyR2; 0.378 ± 0.086 in 150 µM FPL, n = 9, P < 0.01) by prolonging open times and decreasing closed times without changing current magnitude. FPL had no effect on P_o when added to the trans (luminal) side of the bilayer (P_o = 0.079 ± 0.036 in control and 0.103 ± 0.066 in FPL, n = 4, no significant difference). The bell-shaped [Ca²⁺] dependence of [³H]ryanodine binding and of P_o was altered by FPL, suggesting that the mechanism by which FPL increases channel activity is by an increase in Ca²⁺-induced activation at low [Ca²⁺] (without a change in threshold) and suppression of Ca²⁺-induced inactivation at high [Ca²⁺]. However, the fact that inactivation was restored at elevated [Ca²⁺] suggests a competitive interaction between Ca²⁺ and FPL on inactivation. FPL had no effect on RyR skeletal channels (RyR1), where P_o was 0.039 ± 0.005 in control versus 0.030 ± 0.006 in 150 µM FPL (no significant difference). These results suggest that, in addition to its ability to activate the L-type Ca²⁺ channels, FPL activates cardiac RyR2 primarily by reducing the Ca²⁺ sensitivity of inactivation.