A set of hybridoma cell lines, namely, L10, B10, C32, B1, E7, E40 and B49, secreting monoclonal antibodies (MABs) specific to F41 adhesin antigens of ETEC was obtained by somatic hybridization. These MABs manifested strongly specific binding reactivities in DA, ELISA and IFA to all tested F41+ ETEC strains. All these MABs recognized the same or closely related epitopes on the adhesin, as indicated in antigen-competition ELISA with HRP-labeled MABs. The distribution patterns of these epitopes which appeared repeatedly on each pilus were visualized by MAB-immunogold technique and immunoelectron microscopy. The adhesion of ETEC bearing F41 adhesin to the epithelial cells of small intestine from new borne calves and piglets was inhibited in vitro by F41-specific MABs. A dot-immunobinding assay based on F41-specific MABs was developed for determining F41 antigens in fecal samples with HRP-labeled MABs. The assay was highly sensitive and specific, and could be used in rapid detection and screening of F41 adhesin antigens in field samples.