The polycationic dyes, Hoechst 33342 (Bisbenzimide,2'-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi-1H- benzimidazole) and Hoechst 33258 (Bisbenzimide,2'-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)- 2,5'-bi-1H-benzimidazole) alter the activity of the sarcoplasmic reticulum Ca2+ channel. Although they act competitively, Hoechst 33342 decreases, while Hoechst 33258 increases, the rate of channel-mediated Ca2+ efflux from junctional sarcoplasmic reticulum vesicles. Unlike other cationic sarcoplasmic reticulum Ca2+ channel antagonists, Hoechst 33342 blocks the ryanodine-activated Ca2+ channel. Both Hoechst 33342 and Hoechst 33258 inhibit the channel incorporated into the planar lipid bilayer. Since the only structural difference between the two dyes is that the agonist Hoechst 33258 has a hydroxy group where the antagonist Hoechst 33342 has an ethoxy group, it is possible that the more hydrophobic, bulky ethoxy group blocks Ca2+ movement through the channel, whereas the hydroxy group only reduces the rate of Ca2+ movement.