Aortic valve infective endocarditis (IE) can be complicated with severe aortic regurgitation (AR) jet-related lesions such as vegetations, pseudoaneurysms, aneurysms or perforations on the anterior mitral leaflet. Herein is reported the case of a 69-year-old male with culture-negative aortic valve endocarditis complicated with aortic valve perforations resulting in severe and eccentric AR and an AR jet-related ventricular septal defect (VSD). Neither transthoracic echocardiography (TTE) nor two-dimensional transesophageal echocardiography (2D-TEE) were unable to clearly discriminate an aorto-right ventricular fistula from a VSD. By contrast, three-dimensional TEE (3D-TEE) demonstrated multiple aortic valve vegetations, aortic valve perforations, severe AR, and an AR jet-related VSD. The 3D-TEE findings were confirmed at surgery, the aortic valve was replaced with a bioprosthetic valve, and the VSD was repaired with a pericardial patch. To the authors' knowledge, this is the first case of a VSD resulting from an AR jet lesion to be detected and characterized using 3D-TEE. The accurate preoperative diagnosis and characterization of the VSD with 3D-TEE contributed to successful surgery and the patient's short- and long-term survival. Video 1A: This four-chamber transthoracic echocardiography (TTE) view with color Doppler shows a color-Doppler jet traversing the basal interventricular septum (IVS) into the right ventricle, predominantly during systole. However, the origin of the jet resulting from an aortic-right ventricular fistula cannot be determined. A mild to moderate degree of tricuspid regurgitation is also noted. Video 1B: Close-up view of the basal IVS demonstrates a color Doppler jet traversing the IVS, predominantly during systole and apparently originating in the left ventricular outflow tract side, as illustrated by a color Doppler acceleration zone. However, and as in Fig. 1A, the origin of the jet resulting from an aortic-right ventricular fistula cannot be determined. Video 1C: Color Doppler two-dimensional TEE view longitudinal to the aortic valve and aortic root, showing a predominantly systolic color Doppler jet originating in the left ventricular outflow tract and/or aorta and crossing the anterior and basal IVS defect into the right ventricle. Videos 1D: These three-dimensional TEE en-face left ventricular outflow tract views of the aortic valve and basal anterior IVS (Video 1D) and medial anterior close up (Video 1E) and lateral en-face (Video 1F) views of the LV outflow tract and IVS clearly define the anterior and basal location of the VSD with irregular borders and a residual mobile tissue fragment. Multiple aortic valve vegetations and a perforation are visible in Video 1D. Videos 1E: These three-dimensional TEE en-face left ventricular outflow tract views of the aortic valve and basal anterior IVS (Video 1D) and medial anterior close up (Video 1E) and lateral en-face (Video 1F) views of the LV outflow tract and IVS clearly define the anterior and basal location of the VSD with irregular borders and a residual mobile tissue fragment. Multiple aortic valve vegetations and a perforation are visible in Video 1D. Videos 1F: These three-dimensional TEE en-face left ventricular outflow tract views of the aortic valve and basal anterior IVS (Video 1D) and medial anterior close up (Video 1E) and lateral en-face (Video 1F) views of the LV outflow tract and IVS clearly define the anterior and basal location of the VSD with irregular borders and a residual mobile tissue fragment. Multiple aortic valve vegetations and a perforation are visible in Video 1D.