The present study compares, from a morphological point of view, the possible differences between perfusion- and immersion-fixation techniques performed on rat myocardium in order to evaluate their reliability. The data we obtained showed that when the two techniques are carried out with particular care, they do not present significant differences in tissue preservation. Furthermore, we also evaluated the ultrastructural of rat myocardium at the end of in vitro experiments. In this case, signs of subcellular changes, such as vacuolar degeneration, swollen mitochondria, intermyofibrillar myelin-like figures and enlargements of non specialized junctions of the intercalated discs can frequently occur. So, our results indicate that the morphological integrity of myocardium is not completely preserved during in vitro investigations.