Rationale: The role played by resident pleural macrophages in the initiation of pleural inflammation is currently unclear. Objective: To evaluate the role of resident pleural macrophages in the initiation of inflammation. Methods: We have utilised a conditional macrophage ablation strategy to determine the role of resident pleural macrophages in the regulation of neutrophil recruitment in a murine model of experimental pleurisy induced by the administration of carrageenan and formalin-fixed Staphylococcus aureus. Measurements and Main Results: Conditional macrophage ablation mice express the human diphtheria toxin receptor under the control of the CD11b promoter such that the administration of diphtheria toxin induces ablation of nearly 97% of resident macrophages. Ablation of resident pleural macrophages prior to the administration of carrageenan or Staphylococcus aureus dramatically reduced neutrophil influx into the pleural cavity. In the carrageenan model, the reduction in neutrophil infiltration was associated with marked early reduction in the level of macrophage inflammatory protein-2 as well as reduced levels of various cytokines including tumor necrosis factor-, interleukin-6 and interleukin-10. Adoptive transfer of non-transgenic macrophages partially restored neutrophil infiltration. We also stimulated macrophage depleted and non-depleted pleural cell populations with carrageenan in vitro and determined the production of chemokines and cytokines. Chemokine and cytokine production was markedly reduced by macrophage depletion reinforcing the role of resident pleural macrophages in the generation of mediators that initiate acute inflammation. Conclusion: These studies indicate a critical role for resident pleural macrophages in sensing perturbation to the local microenvironment and orchestrating subsequent neutrophil infiltration.