The reactive disulfide 4,4'-dithiodipyridine (4,4' DTDP) was added to single cardiac ryanodine receptors (RyRs) in lipid bilayers. The activity of native RyRs, with cytoplasmic (cis) [Ca2+] of 10(-7) M (in the absence of Mg2+ and ATP), increased within approximately 1 min of addition of 1 mM 4,4'-DTDP, and then irreversibly ceased 5 to 6 min after the addition. Channels, inhibited by either 1 mM cis Mg2+ (10(-7) M cis Ca2+) or by 10 mM cis Mg2+ (10(-3) M cis Ca2+), or activated by 4 mM ATP (10(-7) M cis Ca2+), also responded to 1 mM cis 4,4'-DTDP with activation and then loss of activity. Po and mean open time (T(o)) of the maximally activated channels were lower in the presence of Mg2+ than in its absence, and the number of openings within the long time constant components of the open time distribution was reduced. In contrast to the reduced activation by 1 mM 4,4'-DTDP in channels inhibited by Mg2+, and the previously reported enhanced activation by 4,4'-DTDP in channels activated by Ca2+ or caffeine (Eager et al., 1997), the activation produced by 1 mM cis 4,4'-DTDP was the same in the presence and absence of ATP. These results suggest that there is a physical interaction between the ATP binding domain of the cardiac RyR and the SH groups whose oxidation leads to channel activation.