The purposes of this study were to determine splenic volumes using three‐dimensional ultrasonography and to compare these measurements with two‐dimensional splenic indices. Fifty‐two healthy volunteers were studied. Two‐dimensional volume measurements were based on length, width, and thickness, and the splenic index was calculated using the standard prolated ellipsoid formula (length x width x thickness x 0.523). Three‐dimensional volume planar measurements were obtained with a slice by slice technique by manually drawing a region of interest around the spleen from one end of the sweep to the opposite end. These measurements were recorded three times by two observers. In addition, in vitro determination of splenic volume was performed using three cadaveric human spleens in a water bath. No statistically significant interobserver or intraobserver variability was present for either two‐dimensional or three dimensional ultrasonography. Three‐dimensional sonographic estimations of planar splenic volumes and ellipsoid splenic volumes were consistently smaller than two‐dimensional sonographic estimations of splenic volumes. Three‐dimensional sonographic splenic volumes calculated in vitro using the planar method were accurate to within 2% of in vitro water displacement volumes. Three‐dimensional ultrasonography is potentially superior to two‐dimensional sonography for evaluation of irregularly shaped objects, such as the spleen, and can provide improved accuracy over that of traditional two‐dimensional techniques.