Growth factor regulation of the cortical actin cytoskeleton is fundamental to a wide variety of cellular processes. The cortical actin-associated protein, cortactin, regulates the formation of dynamic actin networks via the actin-related protein (Arp)2/3 complex and hence is a key mediator of such responses. In order to reveal novel roles for this versatile protein, we used a proteomics-based approach to isolate cortactin-interacting proteins. This identified several proteins, including CD2-associated protein (CD2AP), as targets for the cortactin Src homology 3 domain. Co-immunoprecipitation of CD2AP with cortactin occurred at endogenous expression levels, was transiently induced by epidermal growth factor (EGF) treatment, and required the cortactin Src homology 3 domain. The CD2AP-binding site for cortactin mapped to the second of three proline-rich regions. Because CD2AP is closely related to Cbl-interacting protein of 85 kDa (CIN85), which regulates growth factor receptor down-regulation via complex formation with Cbl and endophilin, we investigated whether the CD2AP-cortactin complex performs a similar function. EGF treatment of cells led to transient association of Cbl and the epidermal growth factor receptor (EGFR) with a constitutive CD2AP-endophilin complex. Cortactin was recruited into this complex with slightly delayed kinetics compared with Cbl and the EGFR. Immunofluorescence analysis revealed that the EGFR, CD2AP, and cortactin co-localized in regions of EGF-induced membrane ruffles. Therefore, by binding both CD2AP and the Arp2/3 complex, cortactin links receptor endocytosis to actin polymerization, which may facilitate the trafficking of internalized growth factor receptors.