1 The skeletal muscle Ca 2 þ release channel, the ryanodine receptor, is activated by the trypanocidal drug suramin via the calmodulin-binding site. As calmodulin activates and inhibits the ryanodine receptor depending on whether Ca 2 þ is absent or present, suramin analogues were screened for inhibition of the ryanodine receptor. 2 Up to 300 mM, the novel suramin analogue, 4,4 0 -(carbonyl-bis(imino-4,1-phenylene-(2,5-benzimidazolylene)carbonylimino))-bis-benzenesulfonic acid disodium salt (NF676) was not able to significantly inhibit the basal [ 3 H]ryanodine binding. However, kinetic analysis of the high affinity [ 3 H]ryanodine binding elucidates a time-dependent increment of inhibition by NF676, which is indicative for an open channel blocker. 3 Moreover, the ryanodine receptor was much more sensitive towards inhibition by NF676 when preactivated with caffeine or the nonhydrolysable ATP analogue, adenylyl-imidodiphosphate. Nonetheless, the suramin activated ryanodine receptor was not susceptible towards high-affinity NF676 inhibition, indicating an allosteric hindrance between the binding sites of suramin and NF676. 4 In the line of this finding, NF676 per se was not capable to elute the purified ryanodine receptor from a calmodulin-Sepharose, but it prevented the elution by suramin. 5 Other than suramin, NF676 did not inhibit the Ca 2 þ ATPase of the sarcoplasmic reticulum. However, suramin-induced Ca 2 þ release from sarcoplasmic reticulum was completely abrogated by preincubation with NF676. 6 Taken together, we conclude from these data that NF676 represents a novel lead compound as a potent use-dependent blocker of the skeletal muscle ryanodine receptor via an allosteric interaction with the suramin-binding site.