Kupffer and endothelial cells of rat liver were purified by recirculating collagenase perfusion, metrizamide gradient, and counterflow elutriation. Every step of the isolation procedure was followed by light microscopy and histochemistry. The final fractions of purified sinusoidal cells were examined in their isolated state by transmission and scanning electron microscopy. The scope of this manuscript is the detailed documentation and discussion of all technical aspects and problems of a rather new isolation technique, as well as the detailed description of the purified sinusoidal cells of the liver by scanning electron microscopy. Broader acceptance of this isolation procedure should lead to the specific characterization of biochemical and immunological functions of these cells and elucidate their pathophysiological significance.