To study the structural components of microvilli of the KATO-III cell, we used anti-villin, -ezrin, and anti-MVM (microvillous membrane prepared against mouse intestinal microvilli) antibodies. Villin and ezrin cross-link actin bundles of microvilli such as those in the small intestine and renal proximal cells. Electromicroscopically, the cytoskeletal core of microvilli of the KATO-III cell was constituted of actin-filament bundles. The anti-villin antibody but not anti-ezrin antibody reacted with the KATO-III cell as demonstrated by FITC-immunofluorescence and PAP-staining. Anti-villin, anti-MVM, but not anti-ezrin antibody, reacted with the KATO-III cell surface and with intracellular materials. Western blot analysis using anti-villin and anti-MVM antibodies revealed proteins of approximately 95 kDa (villin), and 15 kDa in the microsomal membrane fractions of KATO-III cells, respectively. Immunocytochemical and confocal laser microscopic studies showed that the cell-surface and the intracellular microcysts of KATO-III cells were preferentially decorated by anti-villin and anti-MVM antibodies. These data suggested that some actin-binding proteins, such as villin, were localized at the cell surface and on some of the intracellular cytoplasmic structures of the KATO-III cell.