Although the cDNA for the cardiac ryanodine receptor (RyR2) has been available for more than 15 years, a thorough characterisation of the recombinant RyR2 has yet to be undertaken. We established a stable inducible HEK293 cell line expressing the full-length rabbit RyR2 cDNA and assessed the functionality of the recombinant RyR2 by single-channel analysis. Sarcoplasmic reticulum (SR) membrane vesicles isolated from sheep hearts obtained from an abattoir or from HEK293 cells were incorporated into planar lipid bilayers for single-channel studies as previously described (1). Similar experiments were performed with purified sheep RyR2 (2). The single-channel conductance of the recombinant RyR2 with Ca2+ as the permeant ion was similar to that of channels isolated from cardiac muscle as was the response of the channel to caffeine, micromolar Ca2+ and ryanodine. Likewise, the activation portion of the dose-response relationship of the recombinant RyR2 to ATP was essentially identical to that previously described for RyR2 isolated from cardiac muscle (1). However, in contrast to the effects observed with RyR2 isolated from cardiac muscle, application of ≥5 mM ATP led to complete inactivation of the recombinant channel (15 out of 16 channels compared with 1 out of 16 sheep RyR2). Similarly, purified sheep RyR2 channels also displayed complete inactivation in the presence of ≥5 mM ATP (4 of 4 channels). These results may be highly significant from a physiological perspective. 5 mM ATP is within the physiological concentration range of cellular ATP (3). However, as inactivation by 5 mM ATP was rarely observed in RyR2 isolated from cardiac muscle, these results raise the possibility that the consistent inactivation observed with the recombinant RyR2 and purified RyR2 could be the result of missing accessory proteins and/or co-factors that protect against ATP-dependent inactivation.