Two new human cholangiocarcinoma (CC) cell lines (CC‐SW‐1 and CC‐LP‐1) were established and maintained in culture for 2 years. Histologically, both original liver tumors were adenocarcinomas, and the cell lines exhibited morphologic eatures of moderately differentiated adenocarcinoma. Immunohistochemistry showed that both cell lines were strongly positive for cytokeratin AEI but negative for carbohydrate tumor‐associated antigen, CA19–9. Ultrastructural analysis of both cell lines showed the presence of tight junctional complexes and focally formed microvilli. Both CC cell lines were tumorigenic in nude mice. Cytogenetic analysis showed that both cell lines expressed highly aneuploid karyotypes with numerous structural and numerical deviations. CC‐SW‐1 was hypodiploid with numerous chromosome losses and structural rearrangements, while CC‐LP‐1 was hyperdiploid and displayed multiple additional chromosomes. Doubling times for the CC‐SW‐1 and CC‐LP‐1 cell lines in the presence of 15% fetal bovine serum were 72 hr and 180 hr, respectively. Growth of the CC‐SW‐1 cell line was significantly stimulated in the presence of insulin, while that of the CC‐LP‐1 cell line was significantly augmented by epidermal growth factor (EGF). In contrast, dexamethasone strongly inhibited proliferation of both cell lines in a dose‐dependent manner. Among various recombinant cytokines examined for effects on growth or surface antigen expression on CC cell lines, only interleukin 1‐β (ILI‐β) strongly inhibited growth of the CC‐LP‐1 cell line, while interferons (IFNs) or tumor necrosis factor‐alpha (TNF‐α) were mildly inhibitory. Both tumor cell lines were resistant to natural killer (NK) cells but sensitive to lymphokine‐activated killer (LAK) cells. Preincubation of tumor cells with IFN‐γ, IFN‐α or TNF‐α significantly decreased the susceptibility of each tumor cell line to lysis by LAK cells, and the change in sensitivity did not correlate with the expression of HLA antigens or intercellular adhesion molecule‐1 (ICAM‐1) on the surface of tumor cells. These 2 CC cell lines are expected to provide valuable information about cell biology of human CC. © 1992 Wiley‐Liss, Inc.