It has been suggested that diadenosine polyphosphates may be physiological regulators of ryanodine receptors (RyR) (Holden et al. 1996) and in line with this proposal, certain diadenosine compounds were shown to stimulate [³H]ryanodine binding to microsomal membrane preparations from rat brain, heart and skeletal muscle. The diadenosine compounds stimulated [³H]ryanodine binding at micromolar concentrations and it was suggested that they act at the ATP binding sites on RyR channels. However, if the diadenosine compounds were to play a physiological role in regulating intracellular Ca²⁺release they would be expected to affect RyR function at the concentrations normally found in cells. We therefore investigated the effects of nanomolar concentrations of P1,P5-diadenosine pentaphosphate (Ap5A) on the function of single sheep cardiac RyR channels incorporated into planar phosphatidylethanolamine lipid bilayers.Native RyR from sheep hearts obtained from an abattoir were incorporated into bilayers under voltage-clamp conditions and single-channel recordings were monitored with Ca²⁺ as the permeant ion as previously described (Chan et al. 2000). Experiments were performed at 22-24 °C with all solutions at pH 7.2. The free Ca²⁺ on the trans-side of the bilayer (corresponding to the SR lumen) was 50 mM. On the cis– (cytosolic) side, free Ca²⁺ was maintained at 10 µM by buffering with EGTA.With 40 µM cytosolic free Ca²⁺ as the sole channel activator, cardiac RyR open probability (Po) was 0.038±0.031 (Mean±S.D.; n=3). With the addition of 1 nM and 10 nM Ap5A to the cytosolic channel side, the Po increased to 0.209±0.061 and 0.353±0.077 (Mean±S.D.; n=3; P<0.05), respectively. These results domonstrate that Ap5A can significantly increase Po at low nanomolar levels. It is possible that the diadenosine compounds may interact with RyR channels at a high affinity site that is distinct from the ATP binding sites. Experiments are currently underway to investigate this hypothesis.