Calcium-induced calcium (Ca2+) release drives cardiac myocyte contraction. Ca2+ release from the sarcoplasmic reticulum is determined by the availability and open probability of ryanodine receptors (RyRs), which are arranged into clusters. Computer modelling suggests that cluster heterogeneity, organisation and the distance to the nearest neighbouring clusters may affect their Ca2+ release properties. Currently, there is little known regarding the ultrastructure of RyR clusters in ventricular and atrial myocytes of large mammals, and how this relates to important inter-chamber differences in the spatiotemporal properties of the calcium transient. Therefore, the aim of this study was to determine if there are differences in RyR cluster spatial properties between the atria and ventricle. Young female adult Welsh Mountain sheep (≈18 months) were euthanised with 200 mg.kg-1 sodium pentobarbital administered intravenously. Left atrial and ventricular myocytes were isolated and immunocytologically labelled for RyR. Photo-switchable fluorescent dyes were used and dye-molecule switching events were acquired over 10,000 frames using the Nikon super resolution N-STORM 4.0 setup. RyR cluster distribution was analysed using a custom written MATLAB programme. Protein abundance was quantified by western blot. Data are presented as mean ± SEM for 27-35 cells from 4 animals for immunocytochemistry and tissue from 8 animals for Western blot with 3 technical repeats. Data were compared using paired t-test where appropriate. In both the atria and ventricle, RyRs were predominantly arranged along the Z-lines with few longitudinal projections. However, there were distinct inter-chamber differences in the size and distance between these clusters. Atrial RyR clusters are both larger by 49.0 ± 24.3% (p<0.05) and closer together, with edge-to-edge distances 51.1 ± 4.2% (p<0.0001) and centroid distances 73.5 ± 4.6% (p<0.0001) of that in the ventricle. Western blot analysis showed that RyR protein abundance does not differ between chambers (p=0.49). The use of super resolution microscopy has shown for the first time inter-chamber differences with respect to RyR distribution. This may account for the spatiotemporal differences of triggered Ca2+ release seen in the atria and ventricle. Future experiments will further characterise the spatial properties of essential calcium handling proteins and relate the findings to functional Ca2+ release.