The purpose of the present study was to determine the impact of NK cells on the development of protective adaptive immunity in response to nasal-pulmonary immunization against mycoplasma. Depletion of NK cells before nasal-pulmonary immunization enhanced resistance to mycoplasma respiratory infection. The effect of NK cells on the generation of protective immunity in lungs was dependent on lymphoid cells, as immunization of either SCID mice or immunocompetent mice depleted of CD4(+) T cells did not demonstrate any increased resistance in the presence or absence of NK cells. The presence of NK cells at the time of nasal-pulmonary immunization modulated mycoplasma-specific cytokine responses in lungs and lower respiratory nodes. In particular, NK cells skewed the mycoplasma-specific T cell cytokine responses in the draining lymph nodes to higher IL-4, IL-13, and IL-17 while lowering IFN-gamma responses. Adoptive transfer of total lung lymphocytes isolated from immunized mice into naive mice led to a significant reduction in the mycoplasma numbers in lungs, and the resistance was greater if cells were obtained from immunized mice that were depleted of NK cells. Similar results were obtained if purified B cells, T cells, or CD4(+) T cells were used. Interestingly, this is the first time that a favorable role of functional CD4(+) T cells in mediating protection in mycoplasma respiratory disease was demonstrated. Thus, NK cells can influence the responses of multiple lymphocyte populations capable of mediating resistance to mycoplasma infection.