We aimed to assess the relationship of the rotational position of the aortic root to its underlying ventricular support, and to the position of the inferior margin of the membranous septum, which serves as a surrogate of the atrioventricular conduction axis. We analyzed 40 normal heart specimens (19 children, 21 adults). The inferior margin of the membranous septum was measured relative to the virtual basal ring. The rotational position of the aortic root was determined by assessing the relationship of the aortic leaflet of the mitral valve to the interleaflet triangle between the non-and left coronary leaflets. The extent of supporting fibrous versus myocardial tissues was measured. We also performed a similar investigation of 30 adult computed tomographic data sets. The median age was 0.25 years (44% male) for children, and 64 years (33% male) for adults. The aortic root was positioned centrally in 22 specimens (55%), rotated counterclockwise in 6 (15%), and clockwise in 12 (30%). In the setting of counterclockwise rotation, 53.4% (median) of the supporting circumference was myocardial, as opposed to 41.4% (median) in those with centrally positioned roots, and 31.9% (median) in those with clockwise rotation (P < 0.0001). The position of the inferior margin of the membranous septum was not associated with the rotational position. Analysis of the 30 adult computed tomographic data sets (median age 66.5 years, 57% male) confirmed the positive relationship between clockwise rotation of the aortic root and an increase in the extent of fibrous as opposed to myocardial support. The rotational position of the aortic root correlates with variation in the extent of its fibrous as opposed to myocardial ventricular support, but not with the position of the inferior margin of the membranous septum relative to the virtual basal ring.