Aim: Primary isolated cardiac myocytes undergo a remodelling process in culture with partial loss of their adult phenotype. This process can be retarded by the addition of Cytochalasin D (Cyto D) to the cell culture medium. In this study we characterise the morphology and function of rat cardiac myocytes cultured up to one week in the absence and presence of Cyto D. Methods: We used confocal microscopy to study fluorescently labelled organelles as well as Ca2+-sparks and global Ca2+ transients. The organelles were visualised by adenoviral-mediated gene transfer of organelle specific fluorescent proteins (FP), or small molecular dyes. The electrophysiological properties were analysed with standard patch clamp approaches. Results: By using GPI-anchored FP, we found that functional T-tubules progressively disappear in a pinch-off manner. This process was largely slowed-down in the presence of Cyto D suggesting an involvement of the actin cytoskeleton in this process. In contrast to the T-tubular system the distribution of the golgi apparatus changed more rapidly in the presence of Cyto D. In freshly isolated cells the FP labeled golgi apparatus showed a dot like structure evenly spread throughout the cell. This distribution changed during the time in culture and we detected an accumulation in the perinuclear regions. In experimental series testing global and local Ca2+ transients as well as the contractile behavior of the cells we have found no significant effects of chronic Cyto D stimulation. Patch clamp experiments supported our finding from above, namely, the reduced disappearance of the T-tubules in the presence of Cyto D. When analysing action-potentials we found a profound and characteristic prolongation of action-potential recovery, indicated by longer ADP50 and ADP70. Conclusion: Our results indicate progressive remodelling of cardiac myocytes in culture. This held true for their morphology and their function. We demonstrated the profound involvement of the actin cytoskeleton in this remodelling process.