We have previously demonstrated that tamoxifen inhibits the growth of human cholangiocarcinoma cells in culture and inhibits tumor growth when cells are injected into nude mice. However, the mechanism of action of tamoxifen remains unknown. Here we demonstrate that tamoxifen and trifluoperazine, both potent calmodulin antagonists, induce apoptosis in vitro, probably acting via the Fas system, in human cholangiocarcinoma cells. Human cholangiocarcinoma cell lines heterogeneously express Fas antigen on their surface. Fas-negative and Fas-positive surface-expressing cells were isolated, cloned, and cultured. Fas antibody, tamoxifen, and trifluoperazine induced dose-dependent apoptosis only in Fas-positive cells; Fas-negative cells were unaffected. Furthermore, apoptosis induced by tamoxifen in Fas-positive cells was blocked by an inhibitory Fas antibody. Tamoxifen was not acting through an anti-estrogenic mechanism, because neither Fas-negative nor Fas-positive cells expressed estrogen receptors and the pure anti-estrogen compound, ICI 182780, did not induce apoptosis in either cell line. Fas-negative cells, but not Fas-positive cells, were able to produce tumors when subcutaneously injected into nude mice. These findings suggest Fas may be a candidate oncogene involved in the pathogenesis of cholangiocarcinoma. Furthermore, the similarity between the pro-apoptotic effects of tamoxifen and trifluoperazine support an underlying molecular mechanism for Fas-mediated apoptosis that involves calmodulin.