OBJECTIVE Epithelial ovarian cancers are considered to arise from neoplastic transformation of the ovarian surface epithelium (OSE). However, the earliest events in ovarian carcinogenesis have not been clearly defined because patients are often diagnosed in the advanced stages and useful in vivo and in vitro experimental systems using human OSE cells are lacking. We aimed to improve the availability of experimental models for the study of human ovarian carcinogenesis. METHODS Subcultured human OSE cells were transfected with SV40 large T antigen. Resulting OSE cell lines were characterized using immunocytochemistry and tested tumorigenicity. RESULTS Six immortalized OSE cell lines were obtained. All cell lines essentially retained the original morphological features of normal OSE cells and showed higher proliferation rates and saturation density. Although they were all nontumorigenic in athymic mice, OSE2b-2 sv cells, which were selected in soft agar from colonies of an SV40 large T antigen-expressing transfectant, OSE2b sv, produced tumors on the peritoneal surface, mesothelium, and diaphragm and induced ascites after being injected intraperitoneally. Solid tumors also grew when mice were inoculated subcutaneously. The tumor cells were formed in a solid sheet arrangement and no evidence of glandular or squamous differentiation was present. They were weakly immunostained with an antibody against cytokeratin, and intercellular junctions resembling attachment devices were ultrastructurally present between cells. The tumors were histologically diagnosed as undifferentiated carcinomas. CONCLUSIONS The established cell lines may provide a model system to investigate the mechanisms of cytogenic and molecular changes from normal OSE cells through the various steps of transformation.