A new human extrahepatic bile duct carcinoma cell line (TFK-1) was established from a surgically resected tumor specimen, which was histologically diagnosed as partly papillary adenocarcinoma and partly differentiated tubular adenocarcinoma. The tumor cells cultured in RPMI-1640 medium supplemented with 10% FBS grew as monolayers showing epithelial-like morphology with a population doubling time of 37 hr during exponential growth at passage 40. Chromosome number was distributed in the range of 72 to 76, with a modal number of 73. Tumor markers (CEA, CA19-9, ST-439, DUPAN-2) were negative in culture supernatant and plasma of SCID mice grafted with TFK-1 cells. Though no point mutation at 12 codon of K-ras was detected, expression of c-erb B-2 product and MUC1 antigen was positive. TFK-1 is the third cell line established from extrahepatic bile duct carcinomas in the world literature, and should provide useful information on various aspects of this type of neoplasm.