Isolated hepatocytes provide a useful whole cell in vitro system for the evaluation of xenobiotic metabolism and toxicity. Several modifications of the two-stage collagenase perfusion technique, originally described by Seglen¹, have been reported for the preparation of hepatocytes. This study seeks to investigate the effect of digestion enzymes on the drug-metabolising activity of isolated rat hepatocytes in suspension. Hepatocytes were isolated from male Sprague Dawley rats (180-220 g) using collagenase type II (C II) as described previously². Modifications of this technique using collagenase A/trypsin inhibitor (C A/TI) and collagenase/dispase (C/D) were also investigated. Hepatocytes prepared using the three different digestion enzymes were characterised based on cell yield, % viability (LDH leakage), cytochrome P450 and reduced glutathione (GSH) content, phase 1 metabolism (7-ethoxycoumarin o-deethylase activity (ECOD)) and phase 2 metabolism (glucuronidation and sulfation of 7-hydroxycoumarin (7HC)). For metabolic studies, incubations were performed over 120 min with a cell density of 2×10⁶ viable cells/ml in Krebs Hepes buffer, pH 7.4, in rotating round bottomed flasks at 37^oC under an atmosphere of 5% CO₂/95% O₂. Table 1 shows the parameters measured on initial isolation. There were no statistical differences in these parameters. However, at a substrate concentration of 100 µM the use of C A/TI results in a significant (p<0.05) increase in the capacity to form 7HC (3.32±0.25 nmol/10⁶cells) compared with C II (1.30±0.23 nmol/10⁶cells) and C/D (1.79±0.55 nmol/10⁶cells). C A/TI also results in a significant (p<0.05) increase in the yield of 7HC glucuronide (94.0±9.5 nmol/10⁶cells) compared with C/D (59.4±5.2 nmol/10⁶cells) at a substrate concentration of 500 µM. These initial observations suggest that the use of trypsin inhibitor in perfusion solutions may be beneficial for the metabolic capacity of isolated rat hepatocytes.