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Purpose There is an excessive demand to develop vaccines against many of the pathogens that infect mucosal tissues or have a mucosal port of entry. Parenteral vaccination may protect in some instances, but usually a mucosal vaccination route is of vital importance as the strongest immune response is obtained at the site of vaccine application and in anatomically adjacent mucosae. The aim of this study was to assess the efficacy of conjunctiva-associated lymphoid tissue (CALT) as a mucosal route of immunization. Methods BALB/c and C57BL/6 mice were immunized via conjunctiva with tetanus toxoid (TTd) as a model antigen. 100 μg/mouse of TTd was applied onto conjunctiva of each eye, together with merthiolate-inactivated B. pertussis, which served as adjuvant. Control mice were immunized subcutaneously with 100 μg/mouse of TTd. Results We found TTd-specific IgG and IgA in tears and sera of both mice strains, in addition to IgG positive TTd-specific cells. There was strong correlation between the amount of TTd-specific antibodies in sera and the presence of TTd-specific B cells in draining lymph nodes. B. pertussis enhanced IgG and IgA immune responses in both mouse strains. T cell activation (increase in CD25 expression and in percentages of CD4+, CD8+, and CD3+ cells) and B cell activation (increase in percentages of CD19+ CD25+ cells) occurred in all mice, but mice immunized with TTd in combination with B. pertussis had the strongest responses. Conclusion Immunization via conjunctiva induced TTd-specific local and systemic immune responses. The strongest immune responses developed in mice that received TTd together with B. pertussis.