Abstract In the accompanying study, we demonstrated that following Ag challenge, membrane (m)IL-5Rα expression is attenuated on bronchoalveolar lavage eosinophils, soluble (s)IL-5Rα is detectable in BAL fluid in the absence of increased steady state levels of sIL-5Rα mRNA, and BAL eosinophils become refractory to IL-5 for ex vivo degranulation. We hypothesized that IL-5 regulates its receptor through proteolytic release of mIL-5Rα, which in turn contributes to the presence of sIL-5Rα. Purified human peripheral blood eosinophils were incubated with IL-5 under various conditions and in the presence of different pharmacological agents. A dose-dependent decrease in mIL-5Rα was accompanied by an increase in sIL-5Rα in the supernatant. IL-5 had no ligand-specific effect on mIL-5Rα or sIL-5Rα mRNA levels. The matrix metalloproteinase-specific inhibitors BB-94 and GM6001 and tissue inhibitor of metalloproteinase-3 partially inhibited IL-5-mediated loss of mIL-5Rα, suggesting that sIL-5Rα may be produced by proteolytic cleavage of mIL-5Rα. IL-5 transiently reduced surface expression of β-chain, but had no effect on the expression of GM-CSFRα. Pretreatment of eosinophils with a dose of IL-5 that down-modulated mIL-5Rα rendered these cells unable to degranulate in response to further IL-5 stimulation, but they were fully responsive to GM-CSF. These findings suggest that IL-5-activated eosinophils may lose mIL-5Rα and release sIL-5Rα in vivo, which may limit IL-5-dependent inflammatory events in diseases such as asthma.