A combination of autoradiography of water-soluble compounds and normal and retrograde perfusions was used to study whether there was a heterogeneity in bile salt transport between zone 1 and zone 3 hepatocytes of the rat and, if so, whether such a heterogeneity was due to the localization of these cells in the liver blood-stream, to intrinsic cellular differences, or to both. When a low dose of [3H]taurocholate was administered under pentobarbital sodium anesthesia in vivo or to normally perfused livers, the label was localized primarily in zone 1; in livers perfused in a retrograde direction, it appeared predominantly in zone 3. High doses of [3H]taurocholate administered in vivo and in normal and retrograde perfusions resulted in a more homogeneous labeling of the acini. The plasma disappearance of [3H]taurocholate was similar in normal and retrograde perfusions, but in the latter biliary excretion occurred at a considerably slower rate. From these results it is concluded that at low doses bile salts are primarily transported by zone 1 cells. Zone 3 cells appear to be able to take up taurocholate with ease, but their biliary excretion is slower compared with zone 1 cells.