Isolation of primary cardiomyocytes can be performed for studies from fetuses and neonates. Compared with rat neonatal cardiomyocytes, mouse neonatal cardiomyocytes are sensitive to enzyme digestion. Therefore, they need to optimize a concentration of collagenase and a time point to collect the heart after birth. In addition, neonatal cardiomyocytes have unique characteristics to be recognized although the preparation steps are not complicated. Therefore, depending on a researcher’s interest and aim, they consider which sources of cardiomyocytes are suitable and convenient, for example, a rat or mouse, as well as a fetus and neonate.