The capacity of airway eosinophils, potentially pertinent to allergic diseases of the upper and lower airways, to function as professional APCs, those specifically able to elicit responses from unprimed, Ag-naive CD4+ T cells has been uncertain. We investigated whether airway eosinophils are capable of initiating naive T cell responses in vivo. Eosinophils, isolated free of other APCs from the spleens of IL-5 transgenic mice, following culture with GM-CSF expressed MHC class II and the costimulatory proteins, CD40, CD80, and CD86. Eosinophils, incubated with OVA Ag in vitro, were instilled intratracheally into wild-type recipient mice that adoptively received iv infusions of OVA Ag-specific CD4+ T cells from OVA TCR transgenic mice. OVA-exposed eosinophils elicited activation (CD69 expression), proliferation (BrdU incorporation), and IL-4, but not IFN-γ, cytokine production by OVA-specific CD4+ T cells in paratracheal lymph nodes (LN). Exposure of eosinophils to lysosomotropic NH 4 Cl, which inhibits Ag processing, blocked each of these eosinophil-mediated activation responses of CD4+ T cells. By three-color fluorescence microscopy, OVA Ag-loaded eosinophil APCs were physically interacting with naive OVA-specific CD4+ T cells in paratracheal LN after eosinophil airway instillation. Thus, recruited luminal airway eosinophils are distinct allergic “inflammatory” professional APCs able to activate primary CD4+ T cell responses in regional LNs.