The F17 antigen from bovine enterotoxigenic Escherichia coli strain (E coli 25KHO9), which adhered to calf intestinal villi, was isolated. An enterotoxin-negative derivative (25KHO9st) was used for further studies. Using an immunogold-labeling technique, the F17 antigen was characterized as a fimbrial protein. Pure fimbriae with a subunit molecular weight of 20,000 were obtained by homogenization and use of a sucrose gradient. The adhesion of E coli 25KHO9st was mediated by the F17 fimbriae, as both F17 antibodies and F17 protein blocked the adhesion of the strain 25KHO9st. The F17 fimbriae were serologically distinct from K88, K99, F41, and 987P fimbriae and did not agglutinate bovine, ovine, guinea pig, human, or chicken erythrocytes. Peptide fingerprint analysis revealed F17 and F(Y) adhesins to be homologous, if not identical.