Septation of the truncus arteriosus of the normal chick embryo heart was surveyed systematically with the light microscope. Tissue from replicate samples at successive periods of development was sectioned within an arbitrary coordinate system based on positional reference points along the external surface of the heart. Correlation of several aspects of tissue morphology within this spatial and temporal reference-frame yielded a new description of tissue associations and kinetics during septation. A stable complex of tissue structures appeared in the downstream, distal truncus at Stage 25 and persisted throughout the septation process. This complex consisted of (1) the cephalic margin of the myocardial sheath, and (2) the adjacent bifurcation of the vascular lumen, linked together by (3) the newly condensed Y-shaped strap of cells forming the aorticopulmonary septum. The apparent motion of this septation-complex toward the ventricle(s), the appearance within the thoracic cavity of the adjacent segments of the aortic arches, and measures of tissue length and width suggested that septation was accompanied, and perhaps initiated, by increased tension along the truncus. The truncal ridges remained upstream from the complex, with mesenchymal condensations beneath the endocardium differentiating into the definitive semilunar valves. Downstream from the bifurcation, mesenchyme in the aortic arch region condensed around the separate lumens to form the smooth muscular tunica media of the great arteries. The epicardium developed in a caudocephalic direction along the heart tube. Vagal innervation approached the heart cephalocaudally. Capillaries formed along the dividing truncus in both directions. Autoradiography following 3H-thymidine labelling demonstrated reduced DNA synthetic activity in the cephalic margin of the myocardium and aorticopulmonary septum, compared with the associated loose mesenchyme.