Mycoplasma can establish latent infections and are associated with arthritis, leukemia, and chronic lung disease. We developed an experimental model using deliberate infection of lung cells with Mycoplasma fermentans. Human lung fibroblasts (HLF) were exposed to live M. fermentans and immune-modulating cytokine release was assessed with and without known inducers of cytokine production. M. fermentans increased IL-6, IL-8/CXCL8, MCP-1/CCL2, and Gro-/CXCL1 production. M. fermentans interacted with TNF- to release more IL-6, CXCL8 and CXCL1 than predicted by the responses to either stimulus alone. The effects of live infection were recapitulated by exposure to M. fermentans-derived macrophage-activating lipopeptide-2 (MALP-2), a TLR-2/TLR-6-specific ligand. The synergistic effect of combined stimuli was more pronounced with prolonged incubations. Pre-exposure to TNF- sensitized the cells to subsequent MALP-2 challenge, but pre-exposure to MALP-2 did not alter the IL-6 response to TNF-. Exposure to M. fermentans or MALP-2 did not enhance nuclear localization, DNA binding or transcriptional activity of NF-B nor did they modulate early NF-B activation in response to TNF-. Application of specific MAP kinase inhibitors of various MAP kinases suggested that p38 and JNK/SAPK were involved in early IL-6 release following TNF- and M. fermentans, respectively. The combined response to M. fermentans and TNF-, however, was uniquely sensitive to delayed application of SP 600125 suggesting that JNK/SAPK contributes to the amplification of IL-6 release. Thus, M. fermentans interacts with stimuli like TNF- to amplify lung cell production of immune-modulating cytokines. The mechanisms accounting for this interaction can now be dissected using this in vitro model.