Ryanodine receptors (RyRs) are calcium release channels in the sarcoplasmic reticulum of ventricular myocytes. They provide the molecular basis of the process of calcium-induced calcium release (CICR) that is critical for cardiac excitation-contraction coupling. When in close proximity, adjacent RyRs can activate each other via CICR so that clustering of RyRs can greatly affect the probability with which calcium trigger signals will activate such clusters. For some time, we have therefore investigated the distribution of RyR clusters in rat ventricular myocytes and quantified the size of RyR clusters as well as inter-cluster distances and the distribution pattern of RyRs within clusters. Recently, using the remarkable localization precision of the super-resolution imaging modality DNA-PAINT we visualized punctate labeling within RyR labeling nanodomains, which we confirmed as single RyRs. RyR positions within sub-plasmalemmal RyR clusters revealed that clusters are organized randomly into irregular clustering patterns leaving significant gaps occupied by accessory or regulatory proteins. DNA-PAINT super-resolution imaging supports a quantitative imaging mode termed qPAINT (or quantitative PAINT). We used qPAINT to precisely quantify RyR cluster size and determined that, on average, peripheral clusters contain 8.8 ± 3.6 RyRs (n = 17 cells, 10 animals). RyRs are spaced within clusters at nearest neighbour distances of 40.1 ± 0.9 nm (n = 1802 clusters), similar to recent measurements using electron tomography but greater than the in vitro RyR packing distances observed in artificial bilayers. We constructed a Monte Carlo model of protein cluster assembly which reproduced the cluster morphologies observed in the DNA-PAINT image data suggesting that RyRs commonly cluster through a random assembly process into irregular arrays containing significant gaps. Using the chromatic aberration-free Exchange PAINT double-labeling approach we determined the stoichiometry of the junctional protein junctophilin-2 (JPH2), measured through a modified qPAINT counting algorithm, which varied from cluster to cluster (0.5-3.5) with a mean of 1.38 ± 0.5 (n = 250 clusters, 2 animals). Our observations provide a basis for biophysical models of RyR cluster activation. We will also present imaging data on RyR phosphorylation within clusters and discuss some implications of this data.