Kupffer cells were found to be distributed over zone 1 (periportal), zone 2 (midzonal), and zone 3 (perivenous) of the rat liver acinus in a ratio of 4:3:2. After pronase digestion of the liver, two purified tractions of Kupffer cells could be obtained by centrifugal elutriation. Liver zone marking with methylene blue prior to cell isolation revealed that one fraction contained Kupffer cells from the periportal area; the other consisted mainly of such cells from the midzonal and perivenous areas. Periportal Kupffer cells were larger and showed higher lysosomal enzyme activities on a per cell basis as compared with midzonal and perivenous Kupffer cells. The immediate uptake of 0.31-micrometer. latex particles during a 2-minute perfusion of the liver was mainly accomplished by periportal Kupffer cells. The higher phagocytic activity of these cells was independent of the direction which latex was flushed through the liver. Seven days after in vivo administration of latex particles, at least 80 per cent of all Kupffer cells contained latex, but again periportal Kupffer cells showed a higher phagocytic activity in that they accumulated a relatively larger number of particles per cell. Latex phagocytosis increased the activity of cathepsin D in all Kupffer cells. Titration with pepstatin revealed that periportal Kupffer cells contained many more cathepsin D molecules than pepstatin revealed that periportal Kupffer cells contained many more cathepsin D molecules than midzonal and perivenous Kupffer cells. Latex particle uptake stimulated an increase in the molecular activity of cathepsin D in periportal cells as well as in the number of cathepsin D molecules in perivenous and midzonal cells. Kupffer cells show a functional heterogeneity that is related to their position in the liver acinus. Kupffer cells with a high endocytic activity, large and heterogeneous lysosomes, and high lysosomal enzyme activities are found in the periportal zone. This zonal heterogeneity can be reduced after phagocytosis of a triggering dose of latex.