The branching geometry of the normal, cholangiographically identifiable human biliary tree was studied with an innovative computer-aided three-dimensional (3D) imaging technique. In addition, a serially sectioned conventional paraffin block from a normal donor liver was used to create and quantitatively study a microscopic 3D image. Finally, a geometric model was developed to estimate the enlargement of biliary surfaces imparted by microvilli. The images created by these techniques could be viewed in stationary modes or rotating around any preselected axis. Approximately 7 (+/-3) intrahepatic duct orders were cholangiographically identified. Computerized measurements of the images from three normal livers suggested that the mean total volume of duct orders 1 to 7 shown in the cholangiograms was 16.6 cm3. The volume of the entire macroscopic duct system was estimated to be between 14 and 24 cm3 (mean, 20.4 cm3), with an internal surface of 336 to 575 cm2 (mean, 398 cm2). A geometric model based on electron micrographs suggested that this surface is magnified approximately 5.5-fold by the presence of microvilli. Volume and surface area (SA) measurements of all ducts in the same orders increased nearly exponentially from the first toward the seventh branching order (i.e., from the hilus toward the periphery of the liver), and probably beyond. The microscopic computerized reconstruction of a septal bile duct with its tributaries also allowed volume measurements; the imaged duct system represented 2.7% of the portal tract volume. The data presented herein may help to better evaluate branching patterns of the biliary tree and, eventually, the quantitative aspects of site-restricted cholangiocyte function and their role in the development of biliary diseases.