Primary hepatocytes are widely used in the study of dynamic events like regulation of gene expression, as they are superior to most cell-lines. However, the culturing of the hepatocytes often results in loss of phenotype, e.g. the expression of the cytochrome p450s (CYP). The present study investigated the impact of serum in the culture medium of porcine primary hepatocytes (PPH) on markers of dedifferentiation as well as the impact on CYP induction. The effects were studied in both freshly isolated primary hepatocytes as well as cryopreserved. The exclusion of serum in the culturing media were not introducing significant dedifferentiation as judged by the gene expression of α-fetoprotein, albumin, glucose-6-phosphatase and the constitutive expression of selected transcription factors and CYP. The induction of CYP2B22 and CYP3A29 by phenobarbital and rifampicin, were greater in hepatocytes cultured without serum. The same were not observed for TCDD induced CYP1A2 expression. In conclusion, PPH cultured under serum free conditions results in little or no dedifferentiation, while being more responsive to known CYP inducers. Hence, it can be suggested that PPH cultured under serum free conditions provides a reliable hepatocyte model to investigate CYP gene regulation.