Recent studies indicate that endocytosis of Eph-ephrin complexes may be one of the mechanisms by which a high affinity cell-cell adhesion is converted to a repulsive interaction. In this study, we show that EphA8 undergoes clathrin-mediated endocytosis upon treatment with ephrin-A5, and that EphA8 is associated tightly with Tiam-1, a Rac-specific guanine nucleotide exchange factor. Analysis of EphA8 deletion mutants revealed that a juxtamembrane region in EphA8 is critically involved in endocytosis of EphA8-ephrinA5 complexes. An EphA8 mutant lacking this juxtamembrane portion was defective for endocytosis with ephrinA5, and also displayed a weak association with Tiam-1. Expression of an endocytosis-defective version of EphA8 resulted in a low level of Rac activity in response to ephrin-A5 stimulation. More importantly, down-regulation of Tiam-1 resulted in inefficient endocytosis of EphA8-ephrinA5 complexes. These results suggest that Tiam-1 plays a role in clathrin-dependent endocytosis of EphA8-ephrinA5 complexes.