Key pointsInositol‐1,4,5‐trisphosphate receptors (IP3Rs) modulate pacemaking in embryonic heart, but their role in adult sinoatrial node (SAN) pacemaking is uncertain.We found that stimulation of IP3Rs accelerates spontaneous pacing rate in isolated mouse SAN cells, whereas inhibition of IP3Rs slows pacing.In atrial‐specific sodium‐calcium exchanger (NCX) knockout (KO) SAN cells, where the Ca2+clock is uncoupled from the membrane clock, IP3R agonists and antagonists modulate the rate of spontaneous Ca2+waves, suggesting that IP3R‐mediated Ca2+release modulates the Ca2+clock.IP3R modulation also regulates Ca2+spark parameters, a reflection of ryanodine receptor open probability, consistent with the effect of IP3signalling on Ca2+clock frequency.Modulation of Ca2+clock frequency by IP3signalling in NCX KO SAN cells demonstrates that the effect is independent of NCX.These findings support development of IP3signalling modulators for regulation of heart rate, particularly in heart failure where IP3Rs are upregulated.AbstractCardiac pacemaking initiated by the sinus node is attributable to the interplay of several membrane currents. These include the depolarizing ‘funny current’ (If) and the sodium‐calcium exchanger current (INCX). The latter is activated by ryanodine receptor (RyR)‐mediated calcium (Ca2+) release from the sarcoplasmic reticulum (SR). Another SR Ca2+release channel, the inositol‐1,4,5‐triphosphate receptor (IP3R), has been implicated in the generation of spontaneous Ca2+release in atrial and ventricular cardiomyocytes. Whether IP3R‐mediated Ca2+release also influences SAN automaticity is controversial, in part due to the confounding influence of periodic Ca2+flux through the sarcolemma accompanying each beat. We took advantage of atrial‐specific sodium–calcium exchanger (NCX) knockout (KO) SAN cells to study the influence of IP3signalling on cardiac pacemaking in a system where periodic intracellular Ca2+cycling persists despite the absence of depolarization or Ca2+flux across the sarcolemma. We recorded confocal line scans of spontaneous Ca2+release in WT and NCX KO SAN cells in the presence or absence of an IP3R blocker (2‐aminoethoxydiphenyl borate, 2‐APB), or during block of IP3production by the phospholipase C inhibitor U73122. 2‐APB and U73122 decreased the frequency of spontaneous Ca2+transients and waves in WT and NCX KO cells, respectively. Alternatively, increased IP3production induced by phenylephrine increased Ca2+transient and wave frequency. We conclude that IP3R‐mediated SR Ca2+flux is crucial for initiating and modulating the RyR‐mediated Ca2+cycling that regulates SAN pacemaking. Our results in NCX KO SAN cells also demonstrate that RyRs, but not NCX, are required for IP3to modulate Ca2+clock frequency.