The type 1 ryanodine receptor (RyR1) from rabbit skeletal muscle displayed two distinct degrees of response to cytoplasmic Ca(2+) [high- and low-open probability (P(o)) channels]. Here, we examined the effects of adenine nucleotides and caffeine on these channels and their modulations by sulfhydryl reagents. High-P(o) channels showed biphasic Ca(2+) dependence and were activated by adenine nucleotides and caffeine. Unexpectedly, low-P(o) channels did not respond to either modulator. The addition of a reducing reagent, dithiothreitol, to the cis side converted the high-P(o) channel to a state similar to that of the low-P(o) channel. Treatment with p-chloromercuriphenylsulfonic acid (pCMPS) transformed low-P(o) channels to a high-P(o) channel-like state with stimulation by beta,gamma-methylene-ATP and caffeine. In experiments under redox control using glutathione buffers, shift of the cis potential toward the oxidative state activated the low-P(o) channel, similar to that of the high-P(o) or the pCMPS-treated channel, whereas reductive changes inactivated the high-P(o) channel. Changes in trans redox potential, in contrast, did not affect channel activity of either channel. In all experiments, channels with higher P(o) were stimulated to a great extent by modulators, but ones with lower P(o) were unresponsive. These results suggest that redox states of critical sulfhydryls located on the cytoplasmic side of the RyR1 may alter both gating properties of the channel and responsiveness to channel modulators.