Approximately 90% of malignant ovarian tumours are epithelial and thought to arise from a single cell layer, the ovarian surface epithelium. In culture, human normal ovarian surface epithelial (OSE) cells have a very limited lifespan before they senesce, rarely progressing beyond 10 population doublings. This has restricted the use of normal OSE cells for studying the biology of ovarian surface epithelium and identifying molecular events that contribute to malignant transformation. We have investigated the conditions for culturing human, normal OSE cells in vitro using modified media. Culturing normal OSE cells in a modified medium (NOSE-CM) supplemented with epidermal growth factor, hydrocortisone, insulin and bovine pituitary extract led to significant improvements in the seeding and cloning efficiencies, overall cell growth and lifespan compared to culturing in a basic, nonsupplemented medium (BM) and previously used media (F-12 K medium and William's medium E). Cells cultured in NOSE-CM underwent, on an average, 19.0 population doublings (95% CI 16.3-21.7); cells cultured in BM underwent 0.43-3.52 population doublings over a similar time period. Growth curves established for different lines indicated that OSE cells continued to grow beyond passage 11 and up to passage 18 in NOSE-CM, but never beyond passage 7 when cultured in BM. It is likely that establishing optimal conditions for the growth of OSE cells in vitro will enable studies of the biological and genetic mechanisms of transformation in epithelial ovarian cancers.