The primary ring, a horseshoe-shaped structure situated between the left and right ventricle and connected superiorly to the atrioventricular canal, is the first specialized fast ventricular conduction pathway in the embryonic heart. It has been first defined immunohistochemically and is characterized as a region of slow myocyte proliferation. Recent studies have shown that it participates in spreading the ventricular electrical activation during stages preceding ventricular septation in the mouse, chick, and rat. Here we demonstrate its presence using optical mapping in chicks between embryonic days (ED) 3-5. We then tested the effects of hemodynamic unloading in the organ culture system upon its functionality. In ED3 hearts cultured without hemodynamic loading for 24 h, we observed a significant decrease in the percentage activated through the primary ring conduction pathway. A morphological examination revealed arrested growth, collapse, and elongation of the outflow tract and disorganized trabeculation. A similar reversal toward more primitive activation patterns was observed with culture between ED4 and ED5. This phenotype was completely rescued with the artificial loading of the ventricles with a droplet of silicone oil. We conclude that an appropriate loading is required during the early phases of the conduction system formation and maturation.