Suramin and structurally related compounds increase not only the open probability (P_o) of ryanodine receptor (RyR) channels but also the single-channel conductance in a unique characteristic manner. In this report, we examine the mechanisms underlying the complex changes to cardiac RyR channel function caused by suramin and the evidence that these changes result from an interaction with calmodulin (CaM) binding sites. In the presence of 100 μM cytosolic Ca²⁺, we demonstrate that suramin exerts a triphasic effect on P_o, indicating the presence of high-, intermediate-, and low-affinity suramin binding sites. The effects of suramin binding to high-affinity sites are Ca²⁺-dependent; P_o is decreased and seems to result from a reduction in the sensitivity of the channel to cytosolic Ca²⁺. We suggest that this site is the CaM inhibition site. Suramin also binds to intermediate-affinity sites that mediate an increase in P_o and an increase in conductance. Cytosolic Ca²⁺ is not an absolute requirement for the effects mediated via intermediate-affinity suramin sites. The suramin-induced increase in P_o and conductance are both concentration-dependent. The correlation between the increase in P_o and increase in conductance indicates that the binding events which produce an increase in P_o also lead to an increase in conductance and, because the effect is concentration-dependent, multiple suramin molecules must bind to produce the maximum effect. The low-affinity suramin binding sites are inhibition sites and mediate a reduction in P_o caused by changes to both open and closed lifetimes.