Kupffer cell (KC)-mediated cytotoxicity against tumor cells is of interest, since the liver is a major site of metastatic growth of primary colorectal cancer. KC isolation methods from rat livers, to study the tumoricidal properties of these cells, are based on perfusion of the liver and are therefore not suitable for human KC isolation from liver biopsies. In view of application to isolate KC from small wedge human liver biopsies, we have developed an isolation procedure for rat KC that does not require perfusion techniques. Liver tissue fragments were incubated with pronase with continuous pH registration and neutralization. KC were subsequently separated from other non-parenchymal cells by Nycodenz gradient centrifugation and purified by counterflow centrifugal elutriation. KC and other non-parenchymal cells were identified by immunophenotyping with a cytoplasmic monoclonal antibody ED1 and by ultrastructural analysis. About 3 x 10(6) KC per gram liver were isolated with a final purity of > 95% without loss of viability. To ensure that functionally competent KC were isolated, we assayed cytotoxicity against CC531 tumor cells in a recent developed cell-mediated MTT assay. Maximum cytotoxicity of KC was approximately 40% at an effector to target ratio of 10. In conclusion our approach seems to be a useful and simple method to isolate KC with good functional properties from rat livers, without the need for perfusion techniques.