Transplanting fetal kidney cells (FKCs) can regenerate kidney. This requires in vitro expansion in cell number to acquire enough cells for transplantation. However, FKCs may change their cellular characteristics during expansion and, thus, may not regenerate kidney tissue upon transplantation. We investigated how cell culture period affects cellular characteristics and in vivo regenerative potential of FKCs. As the passage number increased, cell growth rate and colony forming ability decreased while senescence and apoptosis increased. To examine in vivo regenerative potential, FKCs cultured through different numbers of passages were implanted into the parenchyma of kidneys of immunodeficient mice using fibrin gel for 4 wk. Histological analyses showed passage-dependent kidney tissue regeneration, and the regeneration was better when cells from lower number of passages were implanted. This result shows that in vitro culture of FKCs significantly affects the cell characteristics and in vivo tissue regenerative potential.