Caveolae are small 50 – 100 nm invaginations found in the membrane of most cells, including cardiac myocytes. We have recently shown that around 250 proteins are resident within cardiac caveolae1 which, given the size of caveolae, suggests there are heterogeneous populations of these nanodomains. Isolation of the caveolae coat complex (CCC) and super-resolution imaging of caveolae have given new insights into the organisation of caveolar proteins (caveolin 1 (cav1) and cavin 1) in HeLa and endothelial cells. However, these complexes have not been described in cardiac caveolae, which also contain the muscle-specific caveolar proteins caveolin 3 (cav 3) and cavin 4. Here, using the latest techniques in quantitative western blotting (Q-WB)2 and super-resolution imaging, alongside established methods to isolate the CCC3, we are beginning to identify how caveolar proteins are organised in cardiac myocytes. Single cardiac myocytes, isolated from male Wistar rats (250-280 g), were homogenised in Laemmli sample buffer for protein analysis, attached to laminin-coated coverslips for imaging or incubated with the cross-linker dithiobis(succinimidyl propionate) (DSP) for isolation of the CCC. Q-WB was performed on a Protein Simple® WB system with the analysis of recombinant protein calibration standards alongside biological samples to permit absolute quantitation of specific protein concentrations (cav 1, 3, cavin 1, 4)2. For imaging cells were fixed in paraformaldehyde, labelled using antibodies against Cav 3 in combination with cavin 1, cavin 2 or cavin 4 and visualised by Airyscan or dSTORM. For CCC isolation, complexes were fixed in cells in situ by incubation with 2 mM DSP for 1 h. Following lysis, samples were run on a sucrose velocity gradient to separate protein complexes based on size3. Cav 1, cav 3 and cavin 1 are expressed at similar levels in cardiac myocytes. However, cavin 4 expression is much lower in these cells (26% of cavin 1, n=3). Airyscan imaging shows that a greater proportion of cav 3 co-localises with cavin 1 (53.4 ± 1.4%) than either cavin 2 (23.8 ± 3.7%) or cavin 4 (20 ± 2.7%) supporting the hypothesis that cavin isoforms may contribute to the heterogeneous caveolar populations (animal n=3, mean ± SEM). At a finer resolution (~40 nm – dSTORM), we observe the cavin distribution is the likely result of the larger and more numerous clusters formed by cavin 1 than cavin 4. The current protocol used to isolate the CCC appears to selectively isolate complexes containing only cav 1 and cavin 1; the presence of the cross-linker DSP does not affect the cav 3 complex size. Whether this is because cav 1 forms a distinct caveolar population or because DSP does not cross-link cav 3 requires further investigation. This is the first description of the heterogeneous population of caveolae in cardiac myocytes. Understanding the protein composition of the different caveolae is essential in understanding the multiple functions that caveolae perform.