We had found several methods of hepatocyte isolation to be inadequate for removing Kupffer and endothelial cells and so developed a method of selectively killing these nonparenchymal cells in hepatocyte cultures using the known selective uptake and toxicity of the protein synthesis inhibitor ricin and its A chain by nonparenchymal cells. Kupffer cells were quantitated with the Ku-1 monoclonal antibody. Endothelial and Kupffer cells were identified by means of fluorescence microscopy after uptake of fluorescent-labeled, acetylated low-density lipoprotein. Freshly isolated hepatocyte suspensions contained 21% & 1.2% (mean 5 S.E.M.) as many nonparenchymal cells as hepatocytes (n = 12), including 7.6% 2 1.0% as many Kupffer cells as hepatocytes (n = 10). In monolayers of hepatocytes maintained up to 48 hr in serum-free medium, 3% to 7% of cells were Ku-1 positive, and 2% to 11% took up fluorescence-labeled, acetylated low-density lipoprotein. Purification of hepatocytes using Percoll densitygradient centrifugation, elutriation rotor or other means was only partially effective. Hepatocyte monolayers in serum-free medium were incubated with various concentrations of ricin A chain for 1 hr, then washed and studied up to 48 hr later. Optimal treatment with 1.0 ng/ml rich A chain resulted in decreased nonparenchymal cells 24 hr later and nearly complete loss of Kupffer and endothelial cells at 48 hr. Hepatocyte morphology and protein synthesis were unchanged. Ricin A chain can be used to eliminate Kupffer and endothelial cells from hepatocyte cultures. (HEPATOLOGY 1994;20:436-444.) Isolated hepatocytes are widely used in studies of hepatocyte biology. Hepatocyte suspensions prepared by