The atrioventricular node (AVN) is critical to normal cardiac conduction and can also take over ventricular pacemaking should the sinoatrial node fail. AVN activity is generated by multiple sarcolemmal ionic currents (Hancox et al., 2003) and is influenced by intracellular calcium cycling (Ridley et al., 2008). There is some evidence for a role of inositol trisphosphate (IP3) receptor activation (IP3-R) in modulating sinoatrial nodal pacemaking (Ju et al., 2011; Kapoor et al., 2015). We have recently reported that a cell-permeant IP3 analogue increases spontaneous action potential (AP) firing rate in rabbit AVN cells (Cheng et al., 2015). The present study was undertaken to investigate in AVN cells: the distribution of IP3 (subtype 2) receptors, the effect of cell permeant IP3 analogue on ICa and IKr and effects of caged IP3 on spontaneous activity. AVN cells were isolated from adult male New Zealand White rabbits in accordance with UK Home Office legislation. Double immunocytochemistry was carried out using IP3-R2 and RyR2 antibodies and imaged using confocal microscopy. Spontaneous APs and currents were recorded using the whole-cell patch clamp method at 37 oC. Data are presented as mean ± SEM. Statistical comparisons were made using a paired t-test, with significance denoted by P<0.05. IP3-R2 labelling was observed in transverse bands inside the cell and at the cell periphery where it was partially co-localised with RyR2 (n=6). Release of caged-IP3 (100 µM in pipette solution) by UV flash photolysis rapidly increased spontaneous AP rate by 32.1±9.5% (n=7; P<0.05). The slope of pacemaker diastolic potential of APs increased from 73.4±17.6 to 110.5±14.0 mV/s (n=7; P<0.05), whilst AP overshoot, maximal diastolic potential and duration at half-maximal repolarization were unaffected (n=7; P>0.05). Application of the cell permeant IP3 analogue Bt3-Ins(145)P3/AM (10 µM) did not significantly affect L-type ICa amplitude (-13.2±2.6 pA/pF and -11.7±1.6 pA/pF at +20 mV in control and 10 µM IP3/AM solutions, respectively; n=6; P>0.05), or IKr tails at -40 mV (2.2±0.3 pA/pF and 2.1±0.2 pA/pF, respectively; recorded following step depolarization to +20 mV, n=6; P>0.05). Collectively, these data provide further evidence of functional IP3-R expression in AVN cells. Modulation of spontaneous AP rate by IP3-R activation is unlikely to involve direct effects on L-type ICa or IKr.