The objective of the present study was to characterise the TR146 cell culture model as an in vitro model of human buccal mucosa with respect to the enzyme activity in the tissues. For this purpose, the contents of aminopeptidase, carboxypeptidase and esterase in homogenate supernatants of the TR146 cell culture model, and human and porcine buccal epithelium were compared. The esterase activity in the intact cell culture model and in the porcine buccal mucosa was compared. Further, the TR146 cell culture model was used to study the permeability rate and metabolism of leu-enkephalin. The activity of the three enzymes in the TR146 homogenate supernatants was in the same range as the activity in homogenate supernatants of human buccal epithelium. In the TR146 cell culture model, the activity of aminopeptidase (13.70+/-2.10 nmol/min per mg protein) was approx. four times the activity of carboxypeptidase (3.73+/-0.53 nmol/min per mg protein), whereas the level of esterase activity was significantly higher (223.39+/-69.82 nmol/min per mg protein). In the TR146 cell culture model, the apical esterase activity was found significantly higher than the basal activity, and found comparable to the porcine buccal mucosa. However, the esterase activity on the serosal side of the porcine buccal mucosa was higher than in the TR146 cell culture model. Approx. 1.5% of leu-enkephalin permeated the TR146 cell layers within 5 h (P(app) 7.38+/-0.83x10(-7) cm/s) and approx. 77% of intact peptide was still present in the donor phase after 5 h. The present study suggests that the TR146 cell culture model is a valuable in vitro model for permeability and metabolism studies with enzymatically labile drugs, such as leu-enkephalin, intended for buccal drug delivery.