Interspecies difference is an important issue in toxicology research. We compared the potential in vitro metabolism of human, porcine and rat hepatocytes over 2 weeks in culture in an organotypical culture model which reflects the in vivo situation. All three species show similar LDH-rates. Albumin measurements showed that rat cells are about twice as active as human and porcine hepatocytes. The ethoxyresorufin-O-deethylase (EROD) activity of the rat hepatocytes is with about 14 mu U/10(6) cells distinctly higher than those of porcine and human cells (1.8 and 0.5 mu U/10(6)cells respectively), furthermore, the activity of the rat EROD increases slightly during the prolonged time in culture, whereas those of porcine and human enzymes slightly decrease. Concerning ethoxycoumarin-O-deethylase (ECOD), the enzyme activities are found to be in three different ranges where rat cells show the highest activity with 66 mu U/10(6) cells, porcine hepatocytes exhibit an activity of about 23 mu U/10(6) cells, and human activity is lowest with 0.7 mu U/10(6)cells. All three species show a similar decreasing trend of ECOD during the period of study. Regarding the biotransformation of testosterome, human and porcine liver cells form three major metabolites whereas rat cells form a mixture of all measured metabolites. Hence, in vitro metabolism using porcine hepatocytes would be much more scientific sense than one using rat hepatocytes since the metabolic pathways are much closer to human metabolism. (C) 2009 Published by Elsevier Ireland Ltd.