DP4 is a 36-residue synthetic peptide that corresponds to the Leu²⁴⁴²-Pro²⁴⁷⁷ region of RyR1 that contains the reported malignant hyperthermia (MH) mutation site. It has been proposed that DP4 disrupts the normal interdomain interactions that stabilize the closed state of the Ca²+ release channel (Yamamoto, T., R. El-Hayek, and N. Ikemoto. 2000. J. Biol. Chem. 275:11618–11625). We have investigated the effects of DP4 on local SR Ca²+ release events (Ca²+ sparks) in saponin-permeabilized frog skeletal muscle fibers using laser scanning confocal microscopy (line-scan mode, 2 ms/line), as well as the effects of DP4 on frog SR vesicles and frog single RyR Ca²+ release channels reconstituted in planar lipid bilayers. DP4 caused a significant increase in Ca²+ spark frequency in muscle fibers. However, the mean values of the amplitude, rise time, spatial half width, and temporal half duration of the Ca²+ sparks, as well as the distribution of these parameters, remained essentially unchanged in the presence of DP4. Thus, DP4 increased the opening rate, but not the open time of the RyR Ca²+ release channel(s) generating the sparks. DP4 also increased [³H]ryanodine binding to SR vesicles isolated from frog and mammalian skeletal muscle, and increased the open probability of frog RyR Ca²+ release channels reconstituted in bilayers, without changing the amplitude of the current through those channels. However, unlike in Ca²+ spark experiments, DP4 produced a pronounced increase in the open time of channels in bilayers. The same peptide with an Arg¹⁷ to Cys¹⁷ replacement (DP4mut), which corresponds to the Arg²⁴⁵⁸-to-Cys²⁴⁵⁸ mutation in MH, did not produce a significant effect on RyR activation in muscle fibers, bilayers, or SR vesicles. Mg²+ dependence experiments conducted with permeabilized muscle fibers indicate that DP4 preferentially binds to partially Mg²+-free RyR(s), thus promoting channel opening and production of Ca²+ sparks.