Abstract Rationale In eosinophils, the activation of the serine-threonine MAP kinases ERK1 and ERK2 is a critical signal transduction event for numerous ligand-induced biological processes including elaboration of lipid mediators, directed migration, adherence and degranulation. Given that several cytokine receptor systems are associated with cholesterol-rich membrane microdomains and that components of the ERK signaling pathway are known to contain a cholesterol-sensing motif, the current studies tested the hypothesis that modulation of the cellular cholesterol content regulates receptor-mediated ERK activation in eosinophils. Methods Eosinophils were incubated with the cholesterol chelating agent methyl-b-cyclodextrin (cdex) and subsequently stimulated with 100pM IL-5 or 100nM fMLP. ERK activation was determined by immunoblotting for phospho-ERK1 and -ERK2 Results 1) Activation of ERK1 and ERK2 by fMLP and IL-5 is attenuated by preincubation of the eosinophils with 1-6 mg/ml cdex (N=5). 2) The IL-5-stimulated tyrosine phosphorylation of the transcription factor STAT5 is unaffected by pretreatment with 6 mg/ml cdex, suggesting the attenuation of signaling by cdex does not affect this Jak-STAT pathway as it does the ERK pathway. 3) ERK1&2 activation is enhanced by exposure of eosinophils to 4 mg/ml cdex supplemented with 4% (w/w) cholesterol (N=4). Conclusions These studies suggest that a cholesterol-sensing component present in eosinophils regulates the activity of ERK1&2 and may therefore contribute to the biological responsiveness of eosinophils in the inflammatory microenvironment. Furthermore, defects or alterations in cholesterol metabolism may contribute to eosinophil-associated inflammatory diseases such as asthma.