A new technique for the high-yield preparation of isolated rat liver parenchymal cells involves continuous recirculating perfusion of the liver in situ, shaking the liver in buffer in vitro, and filtration through nylon mesh. This method results in about 50% of the liver being converted into intact, isolated parenchymal cells. 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 show the isolated cells are viable, respiring in calcium-containing medium, synthesizing glucose from lactate, being impermeable to inulin, not staining with trypan blue, and retaining structural integrity. Electron microscopy reveals desmosomes are quickly cleaved, hemidesmosomes invaginate and migrate, while tight and gap junctions persist. Cells without these junctions show vacuolization and potassium loss. The isolated cells are comparable to normal hepatic cells in appearance and function. The method involves perfusion with enzyme medium, followed by shaking and filtration. The cells are then examined for morphology and biochemistry, showing they retain most of their structure and function. The technique allows for high-yield preparation of intact cells, which are useful for metabolic studies. The cells are viable, respiring, and capable of glucose synthesis from lactate. The method is efficient and produces cells suitable for various studies, including metabolic and morphological analyses. The study highlights the effectiveness of the new technique in producing intact, functional liver cells for research purposes.A new technique for the high-yield preparation of isolated rat liver parenchymal cells involves continuous recirculating perfusion of the liver in situ, shaking the liver in buffer in vitro, and filtration through nylon mesh. This method results in about 50% of the liver being converted into intact, isolated parenchymal cells. 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 show the isolated cells are viable, respiring in calcium-containing medium, synthesizing glucose from lactate, being impermeable to inulin, not staining with trypan blue, and retaining structural integrity. Electron microscopy reveals desmosomes are quickly cleaved, hemidesmosomes invaginate and migrate, while tight and gap junctions persist. Cells without these junctions show vacuolization and potassium loss. The isolated cells are comparable to normal hepatic cells in appearance and function. The method involves perfusion with enzyme medium, followed by shaking and filtration. The cells are then examined for morphology and biochemistry, showing they retain most of their structure and function. The technique allows for high-yield preparation of intact cells, which are useful for metabolic studies. The cells are viable, respiring, and capable of glucose synthesis from lactate. The method is efficient and produces cells suitable for various studies, including metabolic and morphological analyses. The study highlights the effectiveness of the new technique in producing intact, functional liver cells for research purposes.