There is no agreement, in the chick, about the number of the endocardial cushions within the outflow tract or their pattern of fusion. Also, little is known of their relative contributions to the formation of the arterial valves, the subpulmonary infundibulum, and the arterial valvar sinuses. As the chick heart is an important model for studying septation of the outflow tract, our objective was to clarify these issues. Normal septation of the outflow tract was studied in a series of 60 staged chick hearts, by using stained whole-mount preparations, serial sections, and scanning electron microscopy. A further six hearts were examined subsequent to hatching. At stage 21, two pairs of endocardial cushions were seen within the developing outflow tract. One pair was positioned proximally, with the other pair located distally. By stage 25, a third distal cushion had developed. This finding was before the appearance of two further, intercalated, endocardial cushions, also distally positioned, which were first seen at stage 29. In the arterial segment, the aortic and pulmonary channels were separated by the structure known as the aortopulmonary septum. The dorsal limb of this septum penetrated the distal dorsal cushion, whereas the ventral limb grew between the remaining two distal cushions, both of which were positioned ventrally. The three distal endocardial cushions, and the two intercalated endocardial cushions, contributed to the formation of the leaflets and sinuses of the arterial roots. The two proximal cushions gave rise to a transient septum, which later became transformed into the muscular component of the subpulmonary infundibulum. Concomitant with these changes, an extracardiac tissue plane was formed which separated this newly formed structure from the sinuses of the aortic root. Our study confirms that three endocardial cushions are positioned distally, and two proximally, within the developing outflow tract of the chick. Key words: heart; chick; developmental biology; scanning electron microscopy; embryology; morphogenesisThere have been numerous descriptions concerning septation of the outflow tract of the heart in different vertebrate species. It remains difficult to correlate the findings between different species, and reports regarding septation within the same species are frequently conflicting. To an extent, these difficulties reflect the formidable task of interpreting three-dimensional relationships in a structure as complex as the developing heart, but some do represent real morphologic differences between species. A firm understanding of these differences remains important, both because of the clinical significance of cardiac malformations (Anderson and Becker, 1992), and the use of animal models to establish the mechanisms of normal and abnormal development. Regardless of the species being studied, there is also considerable confusion regarding the nomenclature of the segments within the outflow tract, and their corresponding cushions.The outflow tract of the heart connects the develop...