A human cholangiocarcinoma cell line, designated as HChol-Y1, was established in a protein-free, chemically defined medium after a very short period of primary culture in 0.1% fetal bovine serum (FBS)-containing medium. The cell line has been propagated in this medium for 2 years. The cells grew as a monolayer and the doubling time was about 52 hours. Addition of FBS did not stimulate cell growth (population-doubling time = 50 hr) or increase saturation density. The cells grown in a protein-free medium secreted small amounts of carcinoembryonic antigen (CEA) and large amounts of carbohydrate antigen (CA) 19/9 (CEA: 12.5 +/- 2.1 ng/10(6) cells/48 hr; CA 19/9: 760 +/- 52 IU/10(6) cells/48 hr); these tumor markers were immunohistochemically demonstrated in HChol-Y1 cells. Addition of FBS slightly stimulated the production of CEA and CA 19/9. The HChol-Y1 cell line was xenotransplantable in athymic nude mice and increased the serum CEA and CA 19/9 levels in the tumor-bearing nude mice. For determination as to whether a human carcinoma cell line can proliferate and secrete CEA and CA 19/9 in synthetic medium without any protein supplements, the cells were cultivated long term (2 yr) in a protein-free, chemically defined medium. When this method of cultivation is used, it is easy to purify these substances from spent medium, because contaminating antigens such as FBS or other substances usually added to cultures are absent.