Secretory and systemic antibody response in mice against enteropathogenic Escherichia coli (EPEC) was evaluated. Groups of mice were immunized with formalin inactivated EPEC 0127:H6 strain by intranasal, peroral, intragastric and intrarectal route, with and without cholera toxin (CT) used as mucosal adjuvant. Mice immunized subcutaneously and a non treated control group were included. Other groups of mice were immunized intranasally with different EPEC strains and a non pathogenic E. coli K12 strain. Antibody response tested by ELISA assay showed that specific anti EPEC 0127:H6 antibodies were induced in serum by intranasal, subcutaneous and intragastric routes. A strong increase of antibody response against EPEC 0127:H6 strain was observed in saliva after intranasal delivery, while a lower response was detected by peroral and intrarectal immunization. Only the intranasal route increased IgA anti EPEC 0127:H6 antibody titers in feces. Specific and cross reactive antibodies to EPEC 0127:H6 were seen in mice immunized intranasally with different EPEC strains. Some control mice showed a background of anti EPEC 0127:H6 antibodies in feces. CT had a negative effect as adjuvant. We showed that nasal mucosa rendered the strongest antibody response in serum and secretions. These results might contribute to optimize the protective effect of enteric vaccines against infections associated to EPEC.