The "Holy Grail" of in vitro toxicology is to develop assay systems that mimic the in vivo situation and hence reduce the need for toxicity tests employing experimental animals. However a major problem to be overcome with cell culture models is the rapid loss of differentiated phenotype that markedly limits extrapolation of results to the whole animal (i.e. human) situation. This limitation is most obvious in the application of hepatocyte cultures to predict pathways of metabolism mediated toxicity and results from the rapid loss of cytochrome P450 content. Here we demonstrate that changes in hepatocyte gene expression (e.g. MAP kinase and NF-kappaB activation) occur very early into the well established hepatocyte isolation procedure employing collagenase suggesting that hepatocytes are undergoing a pro-inflammatory ('acute phase') response before they are cultured. Data is presented indicating that the stimulus is, in part, due to oxidative stress but the demonstration of endotoxins in collagenase preparations is likely to exacerbate the situation. Thus appreciation of these early events during hepatocyte isolation represents the surest foundation for the successful application of cultured hepatocytes to toxicology rather than relying on traditional manipulations of hepatocyte culture medium/substratum once differentiated phenotype has already been lost.