Hypersensitivity pneumonitis (HP) is an interstitial lung disease that develops following repeated exposure to environmental antigens. The disease results in alveolitis, granuloma formation and may progress to a fibrotic chronic form, which is associated with significant morbidity and mortality. The severity of the disease correlates with a neutrophil rich influx and an IL-17 response. We used the Saccharopolysporarectivirgula (SR) model of HP to determine whether Toll-like receptors (TLR) 2 and 9 cooperate in neutrophil recruitment and IL-17-associated cytokine production during the development of HP. Stimulation of bone marrow derived macrophages (BMDMs) from C57BL/6, MyD88(-/-) and TLR2/9(-/-) mice with SR demonstrate that SR is a strong inducer of neutrophil chemokines and growth factors. The cytokines induced by SR were MyD88-dependent and, of those, most were partially or completely dependent on TLRs 2 and 9. Following in vivo exposure to SR, CXCL2 production and neutrophil recruitment were reduced in TLR2(-/-) and TLR2/9(-/-) mice suggesting that the response was largely dependent on TLR2; however the reduction was greatest in the TLR2/9(-/-) double knockout mice indicating TLR9 may also contribute to the response. There was a reduction in the levels of pro-inflammatory cytokines TNFα and IL-6 as well as CCL3 and CCL4 in the BALF from TLR2/9(-/-) mice compared to WT and single knockout (SKO) mice exposed one time to SR. The decrease in neutrophil recruitment and TNFα production in the TLR2/9(-/-) mice was maintained throughout 3 weeks of SR exposures in comparison to WT and SKO mice. Both TLRs 2 and 9 contributed to the Th17 response; there was a decrease in Th17 cells and IL-17 mRNA in the TLR2/9(-/-) mice in comparison to the WT and SKO mice. Despite the effects on neutrophil recruitment and the IL-17 response, TLR2/9(-/-) mice developed granuloma formation similarly to WT and SKO mice suggesting that there are additional mediators and pattern recognition receptors involved in the disease.