The ascites of a 78-yr-old Japanese woman with cholangiocarcinoma was used for a primary culture. An established new cell line (designated TK from the Japanese description of cholangiocarcinoma; Tankan-gann) showed conspicuous tumor marker production. A high level of circulating serum tumor markers; carbohydrate antigen (CA) 19-9, 32,000 U/ml; CA50, 6900 U/ml; and carcinoembryonic antigen (CEA), 300 ng/ml (on an average from 10⁶ cells/ml for 3 d culture) were detected in the tissue culture supernatant. With an inoculum of 2 × 10⁷ TK cells, nude mice progressively developed tumors. The histological features of the tumors forming in nude mice showed well-differentiated adenocarcinoma. Within the tumor mass, large amounts of extra cellular fluid retained approximately 590,000 U/ml of CA19-9, 200,000 U/ml of CA50, and 2000 ng/ml of CEA. Alpha-feto–protein was undetectable in the TK culture supernatant. There are few cholangiocarcinoma cell lines producing stable human tumor markers. Newly established TK cells derived from cholangiocarcinoma showed a stable production of serum tumor markers in vivo and in vitro. The changes in the tumor marker secretion ratios were shown to be dependent upon type of tumor cells, i.e., whether they are in vitro or in vivo. These features make TK cells a valuable tool for studying tumor markers.