A number of biochemical and structural differences have been reported between atrial and ventricular tissue. Notably atrial myocytes have limited or no t-tubules, smaller volume and lower ratio of the sarcoplasmic reticulum calcium ATPase (SERCA) to its inhibitory peptide phospholamban. Since excitation contraction coupling in the ventricle is largely initiated in the t-tubule regions and that sarcoplasmic reticulum (SR) calcium content regulates systolic calcium this study aimed to characterise any alterations in intracellular calcium homeostasis between atrial and ventricular myocytes. All experiments were performed on freshly isolated single cardiac myocytes obtained from the left ventricular mid-myocardium or left atrium using an enzymatic method. Sheep were sacrificed humanely by the intravenous administration of pentobarbitone sodium (200mg/kg). Fluo-5F was used to measure intracellular calcium. All experiments were performed at 37^oC and used the perforated patch voltage clamp technique. SR calcium content was quantified by discharging the SR calcium store with caffeine (10mmol/L) and integrating the resulting sodium-calcium exchange current. Data are presented as mean ± SEM. Statistical significance was determined using a students t-tests. Ventricular myocytes were 9% longer and 59% wider than atrial cells (p<0.05, n = 214-327). Peak L-type calcium current was smaller in atrial cells (3.3 ± 0.7 vs 2.3 ± 0.2 pA/pF n = 10-39, p<0.05). However, both the amplitude of the systolic calcium transient (F/F0, 0.8 ± 0.1 vs 1.5 ±0.1, p<0.05, n = 9-35 cells) and the rate of decay of the systolic transient (5.3 ± 0.4 vs 8.2 ± 0.3 s^-1, n = 8-36 <0.001) were increased in atrial cells. The SERCA mediated calcium removal rate was calculated by subtracting the rate of decay of the caffeine evoked calcium transient from that of the systolic calcium transient and was accelerated in atrial cells (4.2 ± 0.6 vs 7.4 ± 0.4 s^-1, n = 7-30, p<0.001). This latter observation is consistent with an increased SR calcium content in atrial cells (56.0 ± 6.8 vs 238.1 ± 17.8 μmol/l, n = 7-28 , p<0.0001). In summary there are substantial differences in intracellular calcium homeostasis between atrial and ventricular myocytes isolated from the sheep. We propose that previously reported alterations in the ratio of SERCA to phospholamban explains the faster SR dependent calcium uptake and SR calcium content in atrial cells. Additionally, the increased SR calcium content provides a possible mechanism for the larger systolic calcium transient in atrial cells despite the smaller L-type calcium current. It remains to be determined if these alterations are important in explaining the higher frequency of atrial arrhythmias observed in clinical practice.