Isolated hepatocytes are one of the most relevant and practical in vitro models for the study of xenobiotic transport and metabolism. Several modifications of the original two-stage collagenase perfusion technique for the preparation of isolated hepatocytes exist. However, there are limited data about the effect of such modifications on xenobiotic transport and metabolism in isolated hepatocytes. The aim of this project is to investigate the effect of the liver digestion enzyme on hepatocyte uptake, metabolism and efflux of xenobiotics. Three different liver digestion enzyme systems were investigated; collagenase type II (CII), collagenase type A with trypsm inhibitor (CA/TI) and a preparation containing collagenase and dispase (C/D).