Primary human renal proximal tubule epithelial cells (RPTECs) are of limited use for basic research and for clinical applications due to their limited lifespan in culture. Here we used two lentivirus vectors carrying the human telomerase (hTERT) and the SV40T antigen (Tag) flanked by loxP sites to reversibly immortalize RPTECs. Transduced RPTEC clones continued to proliferate while retaining biochemical and functional characteristics of primary cells. The clones exhibited contact-inhibited, anchorage- and growth factor-dependent growth and did not form tumors in nude mice, suggesting that the cells were not transformed. Transient Cre expression in these cells led to efficient proviral deletion, upregulation of some renal specific activities, and decreased growth rates. Ultimately, the cells underwent replicative senescence, indicating intact cell cycle control. Thus, reversible immortalization allows the expansion of human RPTECs, leading to large production of RPTECs that retain most tissue-specific properties.