Protective antigens present in whole cells of bovine enterotoxigenic Escherichia coli strains were tested in an infant mouse diarrhea model. Reduction of mortality rates of infant mice suckling vaccinated mothers was measured 1, 2, and 5 days after oral challenge with strain B41 (O101:K99,F41:H-). Vaccines consisted of strains of serogroup O101, O9, O8, or O20 and of variants bearing or not bearing antigens K99 and F41. K99 and F41 expressions were checked by slide agglutination with K99 and F41 antisera and by mannose-resistant microhemagglutination with horse, sheep, and guinea pig erythrocytes. Absence of production of K99 antibodies following hyperimmunization with K99-negative variants was established in rabbit antisera. Strains bearing K99 alone induced less protection of infant mice 5 days after challenge than strains bearing F41 alone or both K99 and F41. Absence of expression of K99 in the vaccinal strains resulted in either a slight decrease (strains bearing additional F41) or complete abolishment (strains bearing K99 alone) of protection. Failure of F41 synthesis by O101 strains or variants resulted in no protection 5 days after challenge. F41 probably also supplied most of the protection induced by the O9 strain. When negative for both K99 and F41, strains of serogroup O101 still provided protection 1 and 2 days after challenge. This protection was also induced by strain H510a, the reference for O101 antigen. Thus, O antigen contributed to the best vaccinal protection, in addition to K99 and F41.