PURPOSE: To evaluate accuracy and interobserver variation of volume measurement of three-dimensional ultrasonography in balloon phantoms similar to gallbladder experimentally. MATERIALS AND METHODS: Two observers measured volume of water-filled balloon phantoms of 10, 30, 50, 70, 90mL, similar to gallbladder, ten times, respectively, using sum of cylinders method of three-dimensional ultrasound. The measurement error and interobserver variation were assessed. RESULTS: The mean of difference between actual and measured volume was less than 5% in all balloon phantoms. The mean difference between actual and measured volume was the largest (4.4%) in 10mL balloon phantom and about 1% in the others. The mean difference between measurements of two observers was about 1% in all balloon phantoms. The standard deviation was the largest (4.4%) in 10mL balloon phantom and decreased according to increasing volume of balloon. CONCLUSION: Three-dimensional ultrasonography was an accurate volume measurement method with acceptable interobserver variation.