Little is known about the genetics and biology of cholangiocarcinoma (intrahepatic bile duct carcinoma). Only three human bile duct carcinoma cell lines have been described in the literature. We present the first detailed cytogenetic analysis of two cell lines; a new cell line designated PCISG231, established in our laboratory, and RPMI-7451, a previously established cell line. Both lines had highly aneuploid karyotypes with complex rearrangements including marker chromosomes. PCISG231, harvested after 50 days in culture, had a modal and median chromosome number of 65, and many cells contained double-minute chromosomes. RPMI-7451 had a modal and median chromosome number of 67. C-banding confirmed the presence of dicentric chromosomes in PCISG231. Q-banding confirmed the absence of the Y chromosome in PCISG231 and the presence of a der(1)t(Y;1)(q11;p11) chromosome in RPMI-7451. Numerical abnormalities common to both lines included trisomies 2, 5, 11, and 20. Chromosomes 1, 5, 7, and 12 were most commonly involved in structural abnormalities in both lines. Consistent chromosomal breakpoints included 7q22 and 12p11-12. PCISG231 was tumorigenic in immunosuppressed nude mice and was histologically similar to the original tumor. Additional cholangiocarcinoma cell lines are being developed to continue the study of the genetics and cell biology of this disorder.