AbstractWe describe the use of a non‐viral, polyethylenimine‐based vector to transfect rat hepatocytes preserved under hypothermic storage. DNA sequences encoding Escherichia coli β‐galactosidase and pea ferredoxin‐NADP(H) oxidoreductase (FNR), cloned into plasmids pCH110 and pKM4 respectively, were used. FNR was detected in the liver of animals transplanted with transfected cells; no reactivity was observed in endogenous parenchyma. The expression of the transgene was transient as it was detectable up to 96 h subsequently declining to undetectable levels. In contrast to non‐transfected cells, the engraftment of FNR‐positive cells was not associated with inflammatory reaction. The percentage of FNR‐positive implanted hepatocytes was at least five times higher than the original transfection efficiency measured in vitro, while the percentage of β‐galactosidase‐positive cells was similar for both methods. These data indicate that the transfection system is effective in the transfer of plasmid DNA into hepatocytes under cold preservation and suggest the advantage of pKM4‐transfected hepatocytes on engraftment in the recipient parenchyma. Copyright © 2005 John Wiley & Sons, Ltd.