Calcium-induced calcium release (CICR), mediated by ryanodine receptors (RyRs), has been shown to affect calcium signals in many cell types including hair cells (Kennedy & Meech, 2002; Lioudyno et al. 2004; Marcotti et al. 2004). The subcellular location of RyRs has been examined in this study to provide further information about the possible role of CICR in the sensory cells of the cochlea of humanely killed rats. We performed post-embedding immunogold labelling with 3 polyclonal and 2 monoclonal antibodies to determine the distribution of RyRs. Labelling was carried out with each antibody on ultrathin sections of post-hearing rat cochleas embedded in two different resins. Cochlear segments were embedded in Lowicryl HM20 resin using a freeze substitution technique and in LR White using a standard ethanolic dehydration procedure. Better preservation of membranes and intracellular structures was obtained with freeze substitution although the LR White embedded tissue retained greater RyR antigenicity. Positive controls carried out on cardiac and skeletal muscle embedded in both resins gave some indication of antibody specificity for the different receptors, RyR1 and RyR2, the former occurring predominantly in skeletal muscle and the latter in the heart. Polyclonal antibodies against RyRs 1, 2 & 3 (kindly provided by Vincenzo Sorrentino, University of Siena, Italy) did not appear to be strictly specific for their respective receptors under our conditions although they did appear specific for ryanodine receptors. Monoclonal antibody 2142 (kindly provided by Tony Lai Cardiff, UK), preferentially labelled RyR1, detecting it 4 times more readily than RyR2 in LR White embedded tissue. Monoclonal antibody C3-33 (Affinity Bioreagents), although RyR specific, did not appear to preferentially label receptor 1 or 2. Labelling was found with all the antibodies on both the inner and outer hair cells. The heaviest labelling in IHCs was found towards the base of the cell. In outer hair cells, labelling was seen throughout the cell, with the heaviest labelling occurring in the region between the nucleus and cuticular plate. Labelling was also associated with the sub-surface cisternae. These findings confirm the presence of RyRs in regions of hair cells where calcium signalling is thought to occur and add to the evidence that CICR plays an important role in calcium regulation in mammalian hair cells.