The aim of this study was to develop three-dimensional (3D) ultrasound imaging of the carotid arteries in order to reduce intraexaminer and interexaminer variabilities of volume measurements in follow-up scans of atherosclerotic plaques. The non-invasive quantification of human atherosclerosis by 3D ultrasound was performed using a three-dimensional computer based image reconstruction. The linear movements of the transducer were driven by a motor triggered by electrocardiography. The method included an option to change angle of insonation and a correction of skin contact adaptation. Repeated measurements were performed at weekly intervals in 70 patients. Variabilities and ranges of three newly developed evaluation procedures of plaque volumes (manual tracing, threshold procedure, watershed algorithm) were used. Intraexaminer variability varied between 2.8% (watershed algorithm) and 4.1% (threshold procedure). Interexaminer variability ranged from 4.2% (manual tracing) to 7.6% (threshold procedure), follow-up measurements from 5.2% (watershed algorithm) to 9.4% (threshold procedure). Pearson correlation coefficients of interexaminer agreement and also those between all three measurement procedures were highly significant (p < 0.01). We conclude that the newly developed 3D computer based image reconstruction allows repeated quantification of moderate atherosclerosis with a high reliability.