Eosinophils are strongly implicated in the pathogenesis of asthma, particularly in damage to the airway epithelial lining. We examined the potential for lactoferrin, a multifunctional glycoprotein present in the airway surface liquid, to activate eosinophils. Incubating eosinophils in tissue culture wells pretreated with 1–100 μg/ml human lactoferrin stimulated concentration-dependent superoxide production by eosinophils. The same concentrations of immobilized transferrin were without effect. The potency of immobilized lactoferrin was approximately one-third that of immobilized secretory IgA in the same experiments. In contrast, immobilized lactoferrin did not stimulate neutrophil superoxide production. Eosinophils bound lactoferrin as determined by flow cytometry and by binding of 125I-labeled lactoferrin. Transferrin did not block binding of 125I-labeled lactoferrin. Soluble lactoferrin, however, did not activate the eosinophils and did not block superoxide production stimulated by immobilized lactoferrin. Immobilized lactoferrin also stimulated release of eosinophil-derived neurotoxin and low levels of leukotriene C4 production; the latter was significantly enhanced in the presence of 100 pg/ml GM-CSF. GM-CSF also enhanced superoxide production and eosinophil-derived neurotoxin release stimulated by the lower concentrations of immobilized lactoferrin. Pretreatment of the lactoferrin with peptide N-glycosidase F or addition of heparin or chondroitin sulfate to the incubation contents had no or only a minimal effect on the activity of immobilized lactoferrin. These results demonstrate that lactoferrin adherent to the surface epithelium may contribute to the activation of eosinophils that infiltrate the airway lumen in eosinophil-associated disorders such as asthma.