[3H]Ryanodine binding to, as well as functions of, ryanodine receptor intracellular Ca2+ release channel complexes are modulated by several adenosine-based compounds. In this study, we determined the effects of endogenous compounds termed diadenosine polyphosphates (ApnAs; n = 2–6 phosphate groups) on [3H]ryanodine binding to membranes prepared from rat brain and skeletal and cardiac muscle. Under low ionic strength buffer conditions, [3H]ryanodine binding to brain membranes was significantly increased by 171% with 333 µMP1,P5-di(adenosine-5′) pentaphosphate (Ap5A) and by 209% with the same concentration of the metabolism-resistant ATP analogue βγ-methyleneadenosine 5′-triphosphate (AMP-PCP) compared with control values for [3H]ryanodine binding of 9.6 ± 1.8 fmol/mg of protein. Dose-related increases in [3H]ryanodine binding were observed for all five ApnAs tested [P1,P2-di(adenosine-5′) pyrophosphate (Ap2A), P1,P3-di(adenosine-5′) triphosphate (Ap3A), P1,P4-di(adenosine-5′) tetraphosphate (Ap4A), Ap5A, and P1,P6-di(adenosine-5′) hexaphosphate (Ap6A)] as well as AMP-PCP; oxidized salts of ApnAs stimulated [3H]ryanodine binding to a greater degree than did nonoxidized ApnAs. The apparent rank order for the capacity of these agents to increase [3H]-ryanodine binding was oxidized Ap4A = oxidized Ap5A > oxidized Ap3A > Ap6A > AMP-PCP > Ap5A > Ap2A. Addition of the approximate EC50 dose of oxidized Ap4A (37 µM) increased the affinity (KD) of ryanodine receptors from 34 ± 7 to 12 ± 2 nM; the apparent binding site density (Bmax) was not significantly different from control values of 107 ± 33 fmol/mg of protein. Increases in [3H]-ryanodine binding by either oxidized Ap4A or nonoxidized Ap5A were not further enhanced by coincubation with AMP-PCP, which suggests a similar site of action for the ApnAs and AMP-PCP. [3H]Ryanodine binding to skeletal and cardiac muscle membranes was enhanced by addition of oxidized Ap4A, Ap5A, and AMP-PCP. Oxidized Ap4A increased the specific binding by ninefold in skeletal muscle and by threefold in cardiac muscle. These results suggest that ApnAs, at physiologically relevant concentrations, may serve as endogenous modulators of ryanodine receptor-gated Ca2+ release channels.