Cell adhesion molecules are essential for development and maintenance of epithelial architecture. To clarify the role of these molecules in the morphology of gallbladder cancers, four human gallbladder cancer cell lines (GB-d1, KMG-C, GBK-1, and G-415) were examined in vitro. They showed noticeably different morphologies in our standard gel cultures (SC). GB-dl and KMG-C formed cystic and spheroid structures, respectively, which seemed to represent well-differentiated and moderately differentiated cancers, respectively. GBK-1 and G-415 showed branching and "pseudoglandular" structures, respectively, both of which seemed to indicate original dedifferentiated cancers. In floating gel culture (FC), only GB-d1 showed a highly increased tendency toward cyst formation. Expression of E-cadherin and alpha-catenin in the gallbladder cancer cell lines was investigated by Western-blotting analysis. Expression was detected in GB-d1 and KMG-C, but not in GBK-1 and G-415 cells. Furthermore, E-cadherin expression in GB-dl was 1.82 times greater in FC than in SC, while E-cadherin expression levels of KMG-C did not change. Neither GB-d1 nor KMG-C showed any difference in a-catenin expression between SC and FC. Immunostaining of GB-d1 revealed that these proteins were localized to the cell membrane. In contrast, heterogeneous localization of these proteins was detected in the spheroid structures of KMG-C, in both SC and FC. Electronmicroscopic examination revealed that reestablishment of the junctional complex occurred only in GB-d1 cells cultured in FC. The formation of cystic structures in GB-d1 was completely inhibited by an antibody against human E-cadherin. Both expression of E-cadherin and its membranous localization are required for well-differentiated-type morphogenesis in gallbladder cancer cells.