Investigation of the membranous portion of the interventricular septum (MPIS) and its relationship with the aortic valve was performed in 32 human hearts of adult individuals (19 Caucasians and 11 non-Caucasians). With transillumination of the MPIS the specimens were photographed and 35 mm slices obtained. These were digitized into Apple Macintosh II using a Dage Model 68 video camera and a Data Translations DT 2255 frame grabber. The areas and the distances were traced manually, using the NIH Image program (Wayne Rasband, NIH, Research Services Branch, NIHM). The following forms of MPIS were found: oval (31.3%), triangular (28.1%), quadrangular (18.8%), circular (15.6%) and semilunar (6.2%); its surface area varied from 5.65 mm2 to 142.63 mm2 (mean 48.82 +/- 29.17 mm2). The superior border of the MPIS was in close relationship with the aortic valve, and its upper part was usually (41%) in direct continuity with the attachments of both right (RAC) and posterior (PAC) aortic cusps, or with PAC (34%) or RAC (6%) only. Rarely (19%) the MPIS lay below the attachments of both cusps. The distance between the MPIS superior border and the attachment of the RAC was not greater than 5.89 mm (mean 1.69 mm +/- 1.9 mm). The distance between the superior border of the MPIS and the attachment of the PAC was not greater than 5.63 mm (mean 0.77 +/- 1.49 mm). Differences between sex, race and age groups were not statistically significant.