The ovarian surface epithelium (OSE) is thought to give rise to over 85% of human ovarian carcinomas. In spite of its clinical importance, no animal models for the in vivo investigation of this tissue exist, and available culture methods have yielded limited success. In this study, OSE cells from 55 normal ovarian biopsy specimens were used to improve and simplify the methodology for OSE culture and to define the influence of clinical parameters on cultured OSE cells. An improved explanation method was developed which takes advantage of the tenuous attachment of OSE to underlying tissues: the surface epithelium was scraped off the ovarian surface with a rubber scraper, generating epithelial fragments which produced monolayers in culture, with little contamination by other cell types. The scrape method is superior to the explant method previously described (Siemens CH, Auersperg N: J Cell Physiol 134:347, 1988) in terms of speed, simplicity, higher purity of cultures, and increased cell yield. An improved nutrient medium (199/MCDB105/15%FBS) resulted in OSE lines that maintained the original epithelial phenotype for up to 12 population doublings. OSE, detached from the ovary, remained viable if frozen in liquid nitrogen either before culture or in primary culture on strips of plastic, providing OSE independently of the availability of surgical specimens. Growth was not influenced by diagnosis (nonmalignant gynecologic disorders), patient age (mean range: 40.5, 20 to 62 years), or the presence of inclusion cysts or large follicles in the biopsy specimen. This culture system provides conditions for in depth studies of OSE physiology and pathology.