A rapid method for mass isolation of functionally intact hepatocytes and reticuloendothelial cells from a single rat liver is described. The technique is based on collagenase perfusion of the liver, isopycnic sedimentation in Percoll, and selective adherence of the cells. The Kupffer cells (KC) attach and spread on glass or plastic in serum-free medium 15 min following seeding. Cultures of KC are 90%-95% pure with about 5% liver endothelial cells (LEC), less than 1% parenchymal cells (PC) and a maximum of 5% stellate cells (SC). The LEC adhere and spread on fibronectin 60-120 min following seeding, forming cultures that are contaminated with 5-10% SC and less than 1% KC and PC. The yield of plated LEC is 50-60 X 10(6) per 200-g rat. Ultrastructural analysis shows that Percoll does not associate with the cells during the separation procedure.