Effects of the reactive disulfides, 2,2'- and 4,4'-dithiodipyridine, on single cardiac ryanodine receptor (RyR) ion channels incorporated into lipid bilayers are reported. RyRs are activated within minutes of addition of the reactive disulfides (10(-7) to 10(-3) M) with an irreversible loss of channel activity after the activation at concentrations > or = 10(-4) M. This activation, followed by loss of activity, is seen over a wide range of cytoplasmic (cis) Ca2+ concentration between 10(-9) and 2 x 10(-2) M and occurs more rapidly with higher reactive disulfide concentrations or when RyRs are initially active at 10(-5) or 10(-3) M Ca2+. The reactive disulfides increase the channel open probability by introducing long components into the open time distributions, increasing the mean channel open time by up to 50-fold. Closed time distributions are not altered by the sulfhydryl reagents. The effects of the reactive disulfides are prevented by the reducing agents dithiothreitol and glutathione (1-10 mM). The results suggest that cysteine residues on the RyR complex can regulate the ion channel gating mechanisms.