Abstract  An elevation of intracellular Ca²⁺ levels as a result of InsP₃ receptor (InsP₃R) activity represents a ubiquitous signalling pathway controlling a wide variety of cellular events. InsP₃R activity is tightly controlled by the levels of the primary ligands, InsP₃, Ca²⁺ and ATP. Importantly, InsP₃Rs are regulated by in a biphasic manner. Ca²⁺ release through all InsP₃R family members is also modulated dramatically by ATP, albeit with sub‐type‐specific properties. To ascertain if a common mechanism can account for ATP and Ca²⁺ regulation of these InsP₃R family members, we examined the effects of [ATP] on the Ca²⁺ dependency of rat InsP₃R‐1 (rInsP₃R‐1) and mouse InsP₃R‐2 (mInsP₃R‐2) activity expressed in DT40‐3KO cells. We used the on‐nucleus patch clamp recording technique with various [ATP], [InsP₃] and [Ca²⁺] in the patch pipette and measured single InsP₃R channel activity in stably transfected DT40 cells. Under identical conditions, at saturating [InsP₃] and [ATP], the activity of rInsP₃R‐1 and mInsP₃R‐2 was essentially identical in terms of single channel conductance, maximal achievable open probability (P_o) and the [Ca²⁺] required for activation and inhibition of activity. However, in contrast to rInsP₃R‐1 at saturating [InsP₃], the activity of mInsP₃R‐2 was unaffected by [ATP]. At lower [InsP₃], ATP had dramatic effects on mInsP₃R‐2 P_o, but unlike the rInsP₃R‐1, this did not occur by altering the relative Ca²⁺ dependency, but by simply increasing the maximally achievable P_o at a particular [InsP₃] and [Ca²⁺]. [InsP₃] did not alter the biphasic regulation of activity by Ca²⁺ in either rInsP₃R‐1 or mInsP₃R‐2. Analysis of the single channel kinetics indicated that Ca²⁺ and ATP modulate the P_o predominately by facilitating extended bursting activity of the channel but the underlying biophysical mechanism appears to be distinct for each receptor. Subtype‐specific regulation of InsP₃R channel activity probably contributes to the fidelity of Ca²⁺ signalling in cells expressing these receptor subtypes.