Azumolene is an analog of dantrolene, the only approved medicine for treatment of malignant hyperthermia (MH). The pharmacological mechanism of these drugs is to inhibit skeletal muscle sarcoplasmic reticulum (SR) Ca2+ release by modulating the activity of the SR ryanodine receptor (RyR) Ca2+ release channel. To investigate the effects of azumolene on SR Ca2+ channel gating within skeletal muscle fibers, we monitored Ca2+ sparks in permeabilized frog skeletal muscle fibers. Application of 0.0001 to 10 microM azumolene suppressed the frequency of spontaneous Ca2+ sparks in a dose-dependent manner (EC50 = 0.25 microM; Hill coefficient = 1.44), but it did not cause systematic dose-dependent effects on the properties of the Ca2+ sparks. These results suggest that azumolene decreases the likelihood of Ca2+ release channel openings that initiate Ca2+ sparks, thereby decreasing spark frequency, but it has little effect on aggregate Ca2+ channel open times during a spark. To assess azumolene inhibition of RyRs activated in a manner analogous to those activated during an MH episode, we applied DP4, a synthetic peptide corresponding to a central region of RyR1 (Leu2442 to Pro2477), which mimics an MH modification. Azumolene also decreased Ca2+ spark frequency in a dose-dependent manner without altering spark properties in the DP4 MH model. We conclude that azumolene suppresses the opening rate but not the open time of RyR Ca2+ release channels within skeletal fibers.