Background: Outflow tract (OFT) septation defects are a common cause of congenital heart disease. Numerous studies have focused on the septation mechanism of the OFT, but have reported inconsistent conclusions. This study, therefore, aimed to investigate the septation of the aortic sac and the OFT in the early embryonic human heart. Methods: Serial sections of 27 human embryonic hearts from Cegie stage (CS) 10 to CS19 were immunohistochemically stained with antibodies against a-smooth muscle actin (a-SMA) and myosin heavy chain. Results: At CS10-CS11, the OFT wall was an exclusively myocardial structure that was continuous with the aortic sac at the margin of the pericardial cavity. From CS 13 onward, the OFT was divided into nonmyocardial and myocardial portions. The cushion formed gradually, and its distal border with the OFT myocardium was consistently maintained. The aortic sac between the fourth and sixth aortic arch arteries was degenerated. At CS16,the α-SMA-positive aortopulmonary septum formed and fused with the two OFT cushions, thus septating the nonmyocardial portion of the OFT into two arteries. At this stage, the cushions were not fused. At CS19,the bilateral cushions were fused to septate the myocardial portion of the OFT. Conclusions: Data suggest that the OFT cushion is formed before the aortopulmonary septum is formed. Thus, the OFT cushion is not derived from the aortopulmonary septum. In addition, the nonmyocardial part of the OFT is septated into the aorta and pulmonary trunk by the aortopulmonary septum, while the main part of the cushion fuses and septates the myocardial portion of the OFT.