This study describes a novel technique for isolating intact rat liver parenchymal cells with high yield and viability. The method involves continuous recirculating perfusion of the rat liver in situ, followed by shaking in buffer, and filtration through nylon mesh. The perfusion media include calcium-free Hanks' solution with collagenase and hyaluronidase, and magnesium and calcium-free Hanks' solution with EDTA. Biochemical and morphological studies demonstrate that the isolated cells are viable, capable of respiration, glucose synthesis from lactate, and maintaining structural integrity. Electron microscopy reveals that desmosomes are cleaved during perfusion, while tight and gap junctions persist on the isolated cells. The technique yields about 50% of the liver into intact parenchymal cells, significantly higher than previous methods, and is suitable for metabolic and respiratory studies.This study describes a novel technique for isolating intact rat liver parenchymal cells with high yield and viability. The method involves continuous recirculating perfusion of the rat liver in situ, followed by shaking in buffer, and filtration through nylon mesh. The perfusion media include calcium-free Hanks' solution with collagenase and hyaluronidase, and magnesium and calcium-free Hanks' solution with EDTA. Biochemical and morphological studies demonstrate that the isolated cells are viable, capable of respiration, glucose synthesis from lactate, and maintaining structural integrity. Electron microscopy reveals that desmosomes are cleaved during perfusion, while tight and gap junctions persist on the isolated cells. The technique yields about 50% of the liver into intact parenchymal cells, significantly higher than previous methods, and is suitable for metabolic and respiratory studies.