Ryanodine receptors (RyRs) are the major ion channels in the sarcoplasmic reticulum responsible for Ca2+ release in muscle cells. Localization of RyRs is therefore critical to our understanding of Ca2+ cycling and Ca2+-dependent processes within ventricular cells. Recently, RyRs were reportedly found in non-classical locations in the middle of the sarcomere, between perinuclear mitochondria and in the inner mitochondrial membrane of cardiac mitochondria. However, for multiple reasons these reports could not be considered conclusive. Therefore, we modified immunogold labeling to visualize the distribution of RyRs in ventricular myocytes. Using antibodies to the voltage-dependent anion channel (i.e. VDAC) or cytochrome c along with our labeling method, we showed that these mitochondrial proteins were appropriately localized to the mitochondrial outer and inner membrane respectively. Immunogold labeling of ultrathin sections of intact and permeabilized ventricular myocytes with antibodies to three types of RyRs confirmed the existence of RyRs between the Z-lines and around the perinuclear mitochondria. However, we did not find any evidence to support localization of RyRs to the mitochondrial inner membrane.