The transient responses of sheep cardiac and rabbit skeletal ryanodine receptors (RyRs) to step changes in membrane potential and cytosolic [Ca2+] were measured. Both cardiac and skeletal RyRs have two voltage-dependent inactivation processes (tau approximately 1-3 s at +40 mV) that operate at opposite voltage extremes. Approximately one-half to two-thirds of RyRs inactivated when the bilayer voltage was stepped either way between positive and negative values. Inactivation was not detected (within 30 s) in RyRs with Po less than 0.2. Inactivation rates increased with intraburst open probability (Po) and in proportion to the probability of a long-lived, RyR open state (P(OL)) RyR inactivation depended on P(OL) and not on the particular activator (Ca2+ (microM), ATP, caffeine, and ryanodine), inhibitor (mM Ca2+ and Mg2+), or gating mode. The activity of one-half to two-thirds of RyRs declined (i.e., the RyRs inactivated) after [Ca2+] steps from subactivating (0.1 microM) to activating (1-100 microM) levels. This was due to the same inactivation mechanism responsible for inactivation after voltage steps. Both forms of inactivation had the same kinetics and similar dependencies on Po and voltage. Moreover, RyRs that failed to inactivate after voltage steps also did not inactivate after [Ca2+] steps. The inactivating response to [Ca2+] steps (0.1-1 microM) was not RyRs "adapting" to steady [Ca2+] after the step, because a subsequent step from 1 to 100 microM failed to reactivate RyRs.