Mycoplasmas cause chronic respiratory diseases in animals and humans, and to date, development of vaccines have been problematic. Using a murine model of mycoplasma pneumonia, lymphocyte responses, specifically T cells, were shown to confer protection as well as promote immunopathology in mycoplasma disease. Because T cells play such a critical role, it is important to define the role of antigen presenting cells (APC) as these cells may influence either exacerbation of mycoplasma disease pathogenesis or enhancement of protective immunity. The roles of APC, such as dendritic cells and/or macrophages, and their ability to modulate adaptive immunity in mycoplasma disease are currently unknown. Therefore, the purpose of this study was to identify individual pulmonary APC populations that may contribute to the activation of T cell responses during mycoplasma disease pathogenesis. The present study indeed demonstrates increasing numbers of CD11c(-) F4/80(+) cells, which contain macrophages, and more mature/activated CD11c(+) F4/80(-) cells, containing DC, in the lungs after infection. CD11c(-) F4/80(+) macrophage-enriched cells and CD11c(+) F4/80(-) dendritic cell-enriched populations showed different patterns of cytokine mRNA expression, supporting the idea that these cells have different impacts on immunity in response to infection. In fact, DC containing CD11c(+) F4/80(-) cell populations from the lungs of infected mice were most capable of stimulating mycoplasma-specific CD4(+) Th cell responses in vitro. In vivo, these CD11c(+)F4/80(-) cells were co-localized with CD4(+) Th cells in inflammatory infiltrates in the lungs of mycoplasma-infected mice. Thus, CD11c(+)F4/80(-) dendritic cells appear to be the major APC population responsible for pulmonary T cell stimulation in mycoplasma-infected mice, and these dendritic cells likely contribute to responses impacting disease pathogenesis.