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Since impaired gallbladder emptying contributes to gallstone formation, the evaluation of gallbladder motility requires accurate methodology. Recently developed 3-dimensional ultrasonography may take into account various gallbladder shapes more accurately than conventional 2-dimensional ultrasonography. Therefore, volumes of water-filled balloons of various sizes were determined in vitro by 2-dimensional ultrasonography with the sum of cylinders method and by 3-dimensional ultrasonography. Also, in 15 gallstone patients and 6 healthy volunteers, fasting gallbladder volumes and postprandial motility were determined by both methods. Volumes of water-filled balloons as measured by both methods correlated strongly with true volumes (R= 0.93 for 2-dimensional and R = 0.98 for 3- dimensional ultrasonography). Gallbladder volumes measured by both methods were also correlated (R = 0.66, P <0.001). In gallstone patients, 3-dimensional ultrasonography yielded smaller gallbladder volumes than 2-dimensional ultrasonography (P = 0.007), but not in healthy subjects. With both methods, gallstone patients exhibited decreased postprandial gallbladder motility compared to healthy subjects. In conclusion, gallbladder volume measurements by 3-dimensional and 2-dimensional ultrasonography are strongly correlated.Nevertheless, in gallstone patients, gallbladder volumes by 3-dimensional ultrasonography tend to be smaller than by 2-dimensional ultrasonography, possibly due to interference of gallstones with the volume measurement.