To examine whether the line dividing the septum into two layers is found consistently by conventional echocardiography and to evaluate functional differences in the right and left side of the septum in terms of wall thickening, strain rate, and strain imaging. In a systematic study in 30 normal subjects, M mode and Doppler myocardial imaging data from the interventricular septum (IVS) were recorded. Velocity curves, regional strain rate, and strain profiles were obtained. Systolic deformation (wall thickening, radial and longitudinal strain rate, and strain) of both sides were assessed. Furthermore, three patients with one sided abnormalities were studied. A bright echo consistently segmented the IVS into a left and right part. In this normal population radial deformation was different for the left and right side of the septum (mean (SD) wall thickening on the left, 49 (46)%, and on the right, 17 (38)%; strain rate on the left, 3.8 (0.6) 1/s, and on the right, 2.1 (1.9) 1/s; strain on the left, 41 (17)%, and on the right, 22 (14)%), whereas longitudinal deformation was found to be similar (strain rate on the left, -2.2 (0.7) 1/s, and on the right, -2.0 (0.6) 1/s; strain on the left, -28 (12)%, and on the right, -25 (12)%). The presented clinical examples show that abnormalities can be strictly limited to one layer. Differential radial deformation and knowledge of fibre architecture showing an abrupt change in the middle of the septum, together with the clinical cases, suggest the septum to be a morphologically and functionally bilayered structure potentially supplied by different coronary arteries.