Although the Ca2+ fluxes underlying excitation-contraction (EC) coupling in ventricular myocytes are well-defined, there is little information regarding Ca2+ homeostasis in atrial cells. We have previously shown that the sarcoplasmic reticulum (SR) plays a greater role in rabbit atrial than ventricular myocytes (Gadeberg et al., 2013), a finding consistent with atrial-ventricular differences in rat cardiac cells (Walden et al., 2009). Here, we compare the steady-state Ca2+ fluxes underlying EC coupling and cytosolic Ca2+ buffering in voltage-clamped rabbit atrial and ventricular myocytes.Animal procedures were approved by local ethics committee and conducted according to UK legislation. Rabbit atrial and ventricular myocytes were isolated by enzymatic digestion. Resting cytosolic [Ca2+] ([Ca2+]rest) was measured in intact isolated cells loaded with indo-1-AM (atrial 79.0±9.7 nM, n=10; ventricular 97.7±8.4 nM, n=10, P>0.05). Membrane currents and intracellular Ca2+ transients ([Ca2+]i) were measured in separate cells using the whole-cell recording technique (holding potential=-80 mV) and a nominally Ca2+-free, K+-rich pipette solution (pH 7.2) containing 100 μM fluo-4. Cells were superfused with Tyrode’s solution containing 1 mM Ca2+ (pH 7.4, 22 °C). [Ca2+]i was calculated from the normalised fluo-4 fluorescence (F/F0) using the mean [Ca2+]rest obtained for the cell type as measured using indo-1 (Cannell et al., 1994). Ca2+ currents (ICa) were activated by voltage-clamp pulses (100 ms) to 0 mV following a 50 ms pre-pulse to inactivate Na+ current. Steady-state was achieved by a series of 10 command pulses (1 Hz). Unloading of SR Ca2+ was achieved by rapid application of 10 mM caffeine. Na+/Ca2+ exchange current (INCX) was measured as the Ni2+-sensitive component of the caffeine-induced current. SR Ca2+ content was measured as the integral of INCX. The buffering power was calculated as the ratio of the SR Ca2+ content to the amplitude of the caffeine-induced [Ca2+]i transient. Mean data were compared by Student’s unpaired t-test and P<0.05 was considered significant.Although mean ICa density was greater in atrial (6.1±0.4 pA/pF, n=4) than in ventricular myocytes (3.8±0.8 pA/pF, n=3, P>0.05), the ICa-activated [Ca2+]i transient was smaller (192±29 vs. 293±24 nM, P<0.05). This was not due to a difference in the steady state SR Ca2+ content (atrial : 62.4±13.1 μM, ventricular: 58.1±7.2 μM, ns; in contrast to the rat, Walden et al., 2009), however buffering power was greater in atrial cells (238±15 vs 139±13, P<0.01). There was no significant difference in EC coupling gain but fractional release was greater in ventricular (44.9±4.6 %) than in atrial myocytes (31.7±2.0 %, P<0.05). These data demonstrate atrial-ventricular differences in Ca2+ homeostasis in the rabbit heart and that these differ from those previously reported in the rat.