Background: Mycoplasma pneumoniae is a respiratory tract pathogen that has been associated with severe exacerbations in patients with chronic asthma. Murine models of infection have recently been established, with disease manifestations similar to those observed in human subjects. Previous studies have suggested that this organism is capable of producing activation of a wide range of immunologic cell types. Objective: We sought to determine whether M pneumoniae can induce mast cell activation in the rodent mast cell line RBL-2H3. Results: After 4 hours of coculture, morphologic changes indicative of activation were observed by means of electron microscopy, and M pneumoniae was identified, by means of immunoelectron microscopy, adhering to mast cell membranes. Coculture of rat basophilic leukemia cells with viable M pneumoniae for 4 hours resulted in net release of β-hexosaminidase and serotonin into the supernatant. Live, but not heat-killed, organisms induced the release of IL-4 protein into the culture supernatant, with a peak at 4 hours. During coculture with M pneumoniae , production of mRNA for IL-4, IL-6, and TNF-⍺ was upregulated after 2 hours and had returned to near baseline by 24 hours after infection. Conclusions: We conclude that viable M pneumoniae induces activation of mast cells with release of granule contents, as well as cytokine production. (J Allergy Clin Immunol 2002;109:470-6.)