A permanent human neoplastic cell line, DO-s, was established from ascites of a patient with a well-differentiated mucinous cyst-adenocarcinoma of the ovary. This cell line grew as vermiform, floating colonies of epithelial cells in culture. The karyotype of DO-s was of a human female; the chromosome number ranged from 54 to 66 with several abnormalities, mainly trisomy. Epithelial-like character was confirmed by transmission electron microscopy and by the presence of cytokeratin. Inoculation of DO-s cells i.p. or s.c. in athymic nude mice resulted in, respectively, ascites and xenografts. Light and electron microscopical analysis of cultured cells and xenografts demonstrated that the cell line was derived of a mucinous adenocarcinoma biopsy. Tumor-associated antigens, cancer antigen 125 (CA 125), human milk fat globulin, and human placental alkaline phosphatase were expressed by cells in culture and in xenografts. Modulation of the antigens, CA 125 and human milk fat globulin, occurred in DO-s cells growing in athymic mice. Biochemical, immunohistochemical, and histochemical analysis showed that more than 50% of the alkaline phosphatase isoenzymes present in DO-s cells had the characteristics of human placental alkaline phosphatase and placental alkaline phosphatase-like alkaline phosphatase (AP), but fractions of intestinal AP and nonspecific AP (bone-liver-kidney) were also present. The expression of AP isoenzymes could be induced by an enhancement of the serum supplement in the culture media, and by dexamethasone, sodium butyrate, and bromodeoxyuridine. This line will be a valuable tool in studying the therapeutic effects of antibodies to tumor-associated antigens or other agents for ovarian cancer.