The epithelial ovarian carcinomas arise in the ovarian surface epithelium (OSE) which is the mesothelial covering of the ovary. Studies of human OSE have been hampered by the small amounts and limited lifespan of this epithelium in culture. OSE cells expressing SV40 large T antigen (Tag) or the HPV genes E6 and E7 have increased growth potentials but lack some of the normal characteristics of OSE. In this study, we used conditional SV40 Tag expression to produce OSE cells with increased proliferative potentials but relatively normal phenotypes. Primary OSE cultures from three women, one of whom had a BRCA1 mutation, were infected with a temperature-sensitive Tag construct (tsTag), and from these, 28 monoclonal and four polyclonal lines were isolated. The effects of temperature changes were examined in two monoclonal and two polyclonal lines. At the permissive temperature (34° C), these cell lines underwent 52–71 population doublings (PD) compared to 15–20 PD for normal OSE. Nuclear SV40-Tag and p53 expression, demonstrated by immunofluorescence, showed that tsTag was uniformly present and biologically active in all lines. At 34° C, culture morphologies ranged from epithelial to mesenchymal. The mean percentage of cells expressing the epithelial differentiation marker, keratin, varied between lines from 20 to 97%. Collagen type III, a mesenchymal marker expressed by OSE in response to explantation into culture, was present in 24–43% of cells. At 39° C, tsTag was inactivated by 2 d while nuclear p53 staining diminished to control levels over 2 wk. Over 3 d, the cells assumed more epithelial morphologies, keratin expression reached 85–100% in all lines and collagen expression increased significantly in two lines. The cultures with the BRCA1 mutation expressed the most keratin and the least collagen III at both temperatures. As indicated by β-galactosidase staining at pH 6.0, changes leading to senescence were initiated at 39° C by 6 h and were present in all cells after 24 h. However, the cells underwent 1–3 population doublings over up to 1 wk before growth arrest and widespread cell death, thus providing an experimental system where large numbers of OSE cells with different genetic backgrounds and growth potentials can be studied without the concurrent influence of Tag.