Long-term cultures of hepatocytes could represent a suitable in vitro model for biotransformation studies of xenobiotics. At present however, no ideal culture system can be proposed since, in all existing models, phenotypic changes occur, affecting selectively some components of phase I and/or phase II xenobiotic metabolism. From the authors' own results and recent studies of several other investigators, carried out on rat hepatocytes, it becomes clear that four groups of factors may affect biotransformation capacity: soluble medium factors, extracellular matrix components, cell-cell interactions and factors affecting replication. For the maintenance of liver-specific functions, it seems of utmost importance that the tridimensional shape of the hepatocytes is kept. Usually, phase II enzymatic activity is better kept than that of phase I. The cytochrome P450 dependent monoxygenases, in particular, are easily lost. Interesting is the observation that co-cultures of rat hepatocytes with rat liver epithelial cells exhibit higher and much better preserved phase I and phase II biotransformation than monocultures. Clearly, further research is needed to improve this promising in vitro model.