Primary human and rat hepatocyte cultures are well established in vitro systems used in toxicological studies. However, while transgenic mouse models provide an opportunity for studying mechanisms of toxicity, mouse primary hepatocyte cultures are less well described. The potential usefulness of a mouse hepatocyte-based in vitro model was assessed in this study by investigating time-dependent competence for xenobiotic metabolism and gene expression profiles. Primary mouse hepatocytes, isolated using 2-step collagenase perfusion, were cultured in a collagen sandwich configuration. Gene expression profiles and the activities of various Cyp450 enzymes were determined after 0 h, 42 h and 90 h in culture. Principal component analysis of gene expression profiles shows that replicates per time point are quite similar. Gene expression levels of most phase I biotransformation enzymes decrease to ca 69 % and 57 % of the original levels at 42 h and 90 h, respectively, while enzyme activities for most of the studied Cyp450s decrease to 59 % and 34 %. The decrease for phase II gene expression is only to 96 % and 92 % of the original levels at 42 h and 90 h, respectively. Pathway analysis reveals initial effects at the level of proteins, external signaling pathways and energy production. Later effects are observed for transcription, translation, membranes and cell cycle related gene sets. These results indicate that the sandwich-cultured primary mouse hepatocyte system is robust and appears to maintain its metabolic competence better as compared to rat hepatocytes.