Structure-function studies were performed upon the canine in situ interventricular septum. In response to both neural and chemical stimulation, the left septal apex generated greater percent change in contractile force, and contracted earlier than did the basal portion. Under positive inotropic stimulation the left septum contracted earlier and more forcefully than the right. Coarctation of the pulmonary artery elicited moderate augmentation in right septal contraction with no change in the left, whereas partial occlusion of the aorta resulted in increased contractile force in both septa with predominance on the left. Stimulation of the peripheral end of the cervical vagosympathetic trunk induced comparable suppression in contractile force of all surfaces of the septum as well as in both right and left epicardial muscle segments. Based upon careful anatomical dissection of fresh specimens, the interventricular septum was found to be comprised of thin right and relatively thick left muscle masses. The septum may be divided into three different structural zones. The cranial portion is small and consists of a very thin membranous region. The muscular septum is divisible into cranial and caudal portions, each demonstrating characteristic contractile behaviors which are exaggerated under neurally and chemically augmented states and which play an important role in regulation of cardiac output.