A human ovarian carcinoma cell line, OTN 14, has been established from malignant ascitic fluid of a patient with a well-differentiated mucinous cystadenocarcinoma of the left ovary. The cell line has been maintained in vitro for 6 months through 23 passages, growing in monolayers as well as in 3-dimensional clusters, with a population doubling time of 28 1/2 hr. The number of chromosomes per cell varied from 67 to 88, with a modal number of 86. Two characteristic marker chromosomes were recognized, consisting of partially deleted chromosome I. With a DNA index of 1.934 the tumour cell line was near tetraploid. The epithelial character of the OTN 14 cells was confirmed by a positive immunofluorescence reaction with monoclonal antibodies (MAbs) against different keratins, and when (immuno)electron microscopy was used, keratin filaments and small junctional complexes were observed. Vimentin was also expressed in these cells, while desmin was not detected. Cultured tumour cells reacted (weakly) positive with MAb OV-TL 3 as a marker for ovarian carcinomas, while reactivity with the anti-ovarian carcinoma MAb OC 125 was limited to a few cells, not permitting the detection of shed CA 125 antigen in the culture supernatant. Cells stained heterogeneously positive for CEA marker BW 431/31, the presence of which was confirmed by detection of CEA shed into the culture medium. The cell line released estradiol at a concentration of 130,000 pmol/L in the culture medium, while no progesterone or dehydroepiandrosterone sulphate were found. Electron microscopical evidence for steroid production was suggested in some cells showing "dense-core" vesicles near the Golgi areas. The OTN 14 tumour cells formed poorly differentiated tumour nodules in nude mice, and metastatic cells were also found in blood capillaries. Cell types with mucinous as well as endocrine characteristics were found.