The separation process of the heart by which two great arteries and two outflow tracts are formed, was studied microscopically in 20 human embryos, ranging from 6 to 28 mm crown-rump length and macroscopically in eight hearts, ranging from 28 weeks of gestation to 80 years of age. The proximal (primary fold) and distal (ventriculo-arterial junction) borderlines of the outlet segment of the embryonic heart are important landmarks in this process. The remarkable, curved and twisted configuration of the ventriculo-arterial junction implies that the position of the arterial orifices, as well as the relative dimensions of the corresponding outflow tracts, are, already in a very early stage, similar to those in the fully developed heart. It furthermore implies that the separation by the aorto-pulmonary septum starts at this level and immediately involves the outlet segment where the two columns of the aorto-pulmonary septum mobilize the myocardium to form the posterior wall of the right ventricular outflow tract, rather than a septum between both outflow tracts. These findings make the morphology of the outflow tract of the normal heart comprehensible from a developmental point of view and throw a new light upon the morphogenesis of outflow tract malformations.