Background: CC chemokine receptor 3 (CCR3) is a major chemokine receptor involved in regulating eosinophil trafficking, and therefore the elucidation of ligand-induced CCR3 events has important implications in understanding the biologic and pathologic properties of eosinophils. After ligand binding to CCR3, cellular signals include stimulatory (ie, calcium mobilization, actin polymerization, shape change, and chemotaxis) and inhibitory (ie, desensitization of the receptor) events. We have previously demonstrated that CCR3 undergoes rapid and prolonged ligand-induced internalization. Objective: Here we explore the role of internalization in downstream cellular processes, including shape change, actin polymerization, calcium mobilization, and desensitization. Methods: Peripheral blood–derived human eosinophils were pretreated with 2 mechanistically distinct inhibitors of internalization, sucrose and phenylarsine oxide, and functional responses were monitored. Results: We first demonstrate that ligand-induced internalization is required for chemokine-induced eosinophil shape change. To define which signaling components upstream of eosinophil shape change required internalization, we next studied the role of internalization in calcium mobilization and actin polymerization. Sucrose and phenylarsine oxide pretreatment inhibited actin polymerization, implicating receptor internalization in this early response. In contrast, calcium mobilization was not inhibited by blockade of internalization. Finally, we were interested in testing the role of internalization in receptor desensitization. We first demonstrated that preincubation with eotaxin induced a dose-dependent desensitization in eotaxin-induced eosinophil transepithelial migration. However, this phenomenon was not inhibited by blockade of internalization. Conclusion: These results establish that CCR3 internalization is critically involved in select eosinophil functional responses (ie, cellular shape change and actin polymerization) but not desensitization and calcium mobilization. (J Allergy Clin Immunol 2003;111:97-105.)