The external part of the eye shares mucosa-associated common characteristics and is an obvious entry site for foreign Ags. We assessed the potential of eyedrop vaccination for effective delivery of vaccines against viral or bacterial infection in mice. Both OVA-specific IgG Ab in serum and IgA Ab in mucosal compartments were induced by eyedrops of OVA with cholera toxin (CT). Eyedrop vaccination of influenza A/PR/8 virus (H1N1) induced both influenza virus-specific systemic and mucosal Ab responses and protected mice completely against respiratory infection with influenza A/PR/8 virus. In addition, eyedrop vaccination of attenuated Salmonella vaccine strains induced LPS-specific Ab and complete protection against oral challenge of virulent Salmonella. Unlike with the intranasal route, eyedrop vaccinations did not redirect administered Ag into the CNS in the presence of CT. When mice were vaccinated by eyedrop, even after the occlusion of tear drainage from eye to nose, Ag-specific systemic IgG and mucosal IgA Abs could be induced effectively. Of note, eyedrops with OVA plus CT induced organogenesis of conjunctiva-associated lymphoid tissue and increased microfold cell-like cells on the conjunctiva-associated lymphoid tissue in the nictitating membrane on conjunctiva, the mucosal side of the external eye. On the basis of these findings, we propose that the eyedrop route is an alternative to mucosal routes for administering vaccines.