This study determined whether respiratory tract immunization protects against mycoplasma infection and compared preferential immunization of the upper respiratory tract (nasal) with upper and lower respiratory tract (nasal-pulmonary) immunization. Small volumes of inoculum preferentially deposited antigen and induced IgA responses in nasal passages. Larger inoculums deposited antigen in both the upper and lower respiratory tracts, generating corresponding IgA responses. Mice were given nasal or nasal-pulmonary immunizations with Mycoplasma pulmonis antigen alone or with cholera toxin (CT), and resistance to infection was determined. Generation of upper respiratory tract immunity reduced mycoplasma infection at this site, but CT was needed to elicit protective responses. In the lower respiratory tract, nasal-pulmonary immunization was most effective, but nasal immunization did confer some protection from pulmonary infection. In contrast, intraperitoneal immunization resulted in little protection. Thus, respiratory tract immunity plays a major role in resisting mycoplasma infection, and it should be considered during vaccine development.