Pulmonary infection with Pseudomonas aeruginosa is characterized by massive airway inflammation, which comprises significant cytokine production. Although mast cells are abundant in the lung and are potent sources of various cytokines, a role of mast cells in P. aeruginosa infection remains undefined, and P. aeruginosa-induced signaling mechanisms in mast cells have not been studied previously. Here we demonstrate that human cord blood-derived mast cells, mouse bone marrow-derived mast cells, and the mouse mast cell line MC/9 produce significant amounts of interleukin 6 (IL-6) in response to P. aeruginosa. This response was accompanied by a stimulation of protein kinase Calpha (PKCalpha) phosphorylation and PKC activity and was significantly blocked by the PKC inhibitors Ro 31-8220 and PKCalpha pseudosubstrate. Interestingly, mast cells treated with P. aeruginosa had reduced protein levels of phosphatase 2A catalytic unit (PP2Ac), which prompted us to determine whether a direct association between PKCalpha and PP2A occurs in mast cells. In mouse bone marrow-derived mast cells and MC/9 cells, as well as in the human mast cell line HMC-1, PP2A coimmunoprecipitated with PKCalpha either using PKCalpha- or PP2Ac-specific antibodies, suggesting that PKCalpha and PP2Ac are physically associated in mast cells. The PP2A inhibitor okadaic acid induced P. aeruginosa-like responses in mast cells including increased PKCalpha phosphorylation, stimulated PKC activity, and augmented IL-6 production, the last being blocked by the PKC inhibitor Ro 31-8220. Finally, okadaic acid potentiated the P. aeruginosa-induced IL-6 production. Collectively, these data provide, to our knowledge, the first evidence of both a direct physical association of PP2A and PKCalpha in mammalian cells and their coinvolvement in regulating mast cell activation in response to P. aeruginosa.