Ephrins and Eph receptors have key roles in regulation of cell migration during development. We found that the RacGAP beta2-chimaerin (chimerin) bound to EphA2 and EphA4 and inactivated Rac1 in response to ephrinA1 stimulation. EphA4 bound to beta2-chimaerin through its kinase domain and promoted binding of Rac1 to beta2-chimaerin. In addition, knockdown of endogenous beta2-chimaerin blocked ephrinA1-induced suppression of cell migration. These results suggest that beta2-chimaerin is activated by EphA receptors and mediates the EphA receptor-dependent regulation of cell migration.