Abstract The purpose of this study was to validate the accuracy and reliability of volume measurements using three-dimensional (3D) endoluminal ultrasound (ELUS) in canine pseudotumor esophageal specimens in vitro. Pseudotumors were created by injecting various volumes of US gel (0.1–1.0 ml) into canine esophageal specimens. A stepping-motor was used to pull either a 9, 12.5 or 20 MHz transducer through the lumen of the specimen at 1.5 mm/s. Images were downloaded to a LIFE computer system for 3D reconstruction. Volume measurements were made by two investigators and compared to spiral CT images. Averaging across all measurements, the average magnitude of error was 8.7% in individual US determinations and 11.9% in CT measures. Volumes estimated from images spaced 0.5 and 1.0 mm apart, from images in the original and reconstructed planes, and from different scan frequencies, produced percentage errors that were not statistically significantly different from each other on ELUS. 3D ELUS can be used accurately and reproducibly to measure tumor volumes with a low mean percent in vitro.