Thioglycollate-elicited peritoneal cells are a common source of macrophages for various in vitro assays, including stimulation with TLR ligands, cell signaling assays, phagocytosis, toxicology studies, and cytokine/chemokine production. The most common method for enrichment of cultured thioglycollate-elicited peritoneal cells is adherence. However, the presence of other cell types in freshly isolated and cultured thioglycollate-elicited peritoneal cells has not been examined. Here, we demonstrate that thioglycollate-elicited peritoneal cavity contains 55-60% nonmacrophage cells, and even after adherence, there are still 12-20% nonmacrophage cells remaining. Excluding macrophages, eosinophils are the major cell type in the freshly elicited cavity (30-40%). Eosinophils are also the major cell type contaminating in vitro cultures of thioglycollate-elicited peritoneal macrophages. Moreover, the contamination of macrophage cultures by eosinophils significantly diminishes activation of p38 MAPK and the serine threonine kinase Akt and production of proinflammatory cytokines in response to LPS stimulation. Taken together, these data suggest that thioglycollate-elicited peritoneal cells are far more heterogeneous than reported previously. Further, a failure to remove contaminating eosinophils may greatly affect the interpretation of results obtained with cultured thioglycollate-elicited macrophages. Thus, our data indicate that future studies intent on accurately assessing cultured macrophage phenotype and activation require depletion of all cocontaminating cells, especially eosinophils.