Carotid atherosclerotic lesions are a major cause of stroke and the identification and quantification of such lesions in patients is important for the development of a better understanding of atherogenesis in high risk populations and for the design of studies to assess treatment efficacy. Our objective was to develop and validate a new three-dimensional ultrasound (3DUS) measurement or phenotype of carotid atherosclerosis, vessel wall volume (VWV), which is a three-dimensional measurement of vessel wall thickness and plaque within the carotid arteries measured in 3DUS images. To assess both intraobserver and interscan variability, 3DUS images were acquired from the right and left carotid arteries of ten subjects with carotid atherosclerosis scanned twice within a period of 2 wk. For both VWV and total plaque volume (TPV), an expert observer performed five measurement trials of all images acquired at baseline scan and 2-wk rescan with a 5-d period between measurement trials for images. Images were re-randomized for each measurement trial and both TPV and VWV were measured by observers who were blinded to subject identification for each time-point measurement. Coefficients of variation (COV) and intraclass correlation coefficients (ICC), for VWV measurements indicated higher intraobserver (scan COV = 4.6% ICC = 0.95, rescan COV = 3.4%, ICC = 0.96) and interscan reproducibility (COV = 5.7%, ICC = 0.85) than TPV measurements (intraobserver variability scan COV = 22.7% ICC = 0.85, rescan COV = 21.1% ICC = 0.88 and interscan variability, COV = 31.1%, ICC = 0.83), although absolute variances for both phenotypes were very similar (VWV = 90 mm3, TPV = 80 mm3).