Introduction: Suspension of isolated hepatocyes is now being used in an increasing number of biochemical investigations, including drug metabolism and toxicity studies. Isolated hepatocyes, retain many of the essential properties of the intact tissue including permeability charactreristics. Material and Methods: In this method at first liver is subjected to a nonrecirculating perfusion with calcium free containing a calcium chelator like EGTA which causes irreversible separation of gap, tight and desmosomal junctions. In the second step, perfusion is continued with a buffer containing collagenase in order to dissolve extracellular matrix. In this step calcium is added back to the medium to ensure maximal enzyme activity. Results: In situ perfusion of rat liver using collagenase very high recovery of intact isolated hepatocyes (more than %90), Approximately 500 millions of intact cells have been obtained from each rat weighing 200g. Conclusion: The availability of these methods to isolate intact viable hepacoyes has provided a system that can be utilized in biochemistry, pharmacology, and toxicology. Suspension of isolated hepatocyes is now frequently used in biochemical studies. This experimental tool has been successfully employed in studies on gluconeogenesis, glycolysis, protein, lipid, fatty acid, and urea syntheses, keton body production, protein metabolism, ethanol oxidation, membrane transport, and response to hormones.