A worldwide shortage of donor livers is a limiting factor of the clinical application of hepatocyte transplantation (HTX). To resolve this issue, we focused on a reversible immortalization system that allows temporary expansion of primary hepatocyte populations by transfer of an oncogene that can be subsequently excised. As a preliminary test toward this goal, we examined the efficacy of Cre/loxP site-specific recombination in a transformed human liver cell line, HepG2. The present study utilized retroviral transfer of a prototypical immortalizing gene, simian virus 40 large T antigen (SV40Tag), flanked by a pair of loxP recombination targets and adenovirus-mediated Cre/loxP recombination. Here we report that complete elimination of the retroviral transferred oncogene was achieved by site-specific recombination using a replication-deficient recombinant adenovirus vector producing Cre recombinase (Ad-Cre).