Aims: Primary isolated cells represent a widely used model to study physiological relevant processes. In cardiomyocytes, isolation and culturing induces largely unknown remodelling processes. We have introduced a culturing method without major alterations of the cells. With our method, dedifferentiation was largely suppressed in terms of overall morphology (shape and cross- striation) and physiology (contractility). The aim of this study was to elucidate how intracellular structures (such as sarcoplasmic reticulum, mitochondria and cytoskeleton) of cultured myocytes were affected by our culturing procedure. Methods: We analysed changes in the structure of cell compartments with confocal fluorescence imaging and fluorescence recovery after photobleaching (FRAP). To probe the cells during one week in culture we used adenoviral- mediated transduction of targeted fluorescent proteins, small molecule dyes and immunocytochemistry. Results: During one week in culture, we found highly reproducible remodelling of the cells. When following the distribution of the mitochondria over culturing time we could detect an increase in average mitochondrial length along the longitudinal axis from 1.8 mm to about 4 mm, while the perpendicular properties remained unchanged. FRAP-experiments revealed association between apparently separated mitochondria by "tunneling" via sub-resolution organelle-tubes. The recovery time of the fluorescent signal after photobleaching increased, supporting our notion of an increased association between mitochondria during the time of culture. Furthermore, a gradual loss of the cross-striation arrangement in the ER/SR was visualised. Conclusion: Primary isolated cardiomyocytes from adult hearts undergo a well-defined, but reproducible subcellular remodelling during optimised long-term culture. Such an improved system is essential to study long-term effects of chronical electrical or hormonal stimulation on cardiac myocytes.