Few model systems exist for the study of injury to human renal proximal tubule epithelium. Optimized differentiated human renal epithelial cell lines with extended in vitro growth potential would provide an alternative model system to primary culture or other available non-human mammalian kidney cell lines. For this purpose, human renal tubule epithelial cells were isolated from normal kidney cortex and exposed in culture to a hybrid immortalizing virus, adenovirus 12-SV40. Cell lines were developed by limiting dilution, and three selected cell lines were screened for growth pattern, production of immortalizing virus, tumorigenicity, and ploidy. Cell lines were also monitored for response to inducer agents and matrix factors and were screened for expression of biochemical properties and differentiation markers of renal epithelium. All three are nonproducers of the immortalizing virus and are nontumorigenic. They grow in monolayer, have intermediate growth kinetics, and express markers of renal proximal tubular epithelium by immunohistochemistry. They also express biochemical properties comparable to other widely used proximal tubular cell lines including LLC-RK1, OK, and HK-2 and comparable to human tubular cells in stable culture. Growth medium containing low levels of fetal calf serum, or epidermal growth factor combined with parathyroid hormone, produced optimal growth characteristics, brush border enzyme expression, biochemical properties, and glucose transport in a selected cell line. The addition of dimethyl sulfoxide allows maintenance in morphologically intact monolayers for prolonged periods. These cell lines should be useful model systems for the study of human renal proximal tubular injury or disease.