The B cell coreceptor CD22 plays an important role in regulating signal transduction via the B cell Ag receptor. Studies have shown that surface expression of CD22 can be modulated in response to binding of ligand (i.e., mAb). Thus, it is possible that alterations in the level of CD22 expression following binding of natural ligand(s) may affect its ability to modulate the Ag receptor signaling threshold at specific points during B cell development and differentiation. Therefore, it is important to delineate the physiologic mechanism by which CD22 expression is controlled. In the current study, yeast two-hybrid analysis was used to demonstrate that CD22 interacts with AP50, the medium chain subunit of the AP-2 complex, via tyrosine-based internalization motifs in its cytoplasmic domain. This interaction was further characterized using yeast two-hybrid analysis revealing that Tyr843 and surrounding amino acids in the cytoplasmic tail of CD22 comprise the primary binding site for AP50. Subsequent studies using transfectant Jurkat cell lines expressing wild-type or mutant forms of CD22 demonstrated that either Tyr843 or Tyr863 is sufficient for mAb-mediated internalization of CD22 and that these motifs are involved in its interaction with the AP-2 complex, as determined by coprecipitation of α-adaptin. Finally, experiments were performed demonstrating that treatment of B cells with either intact anti-Ig Ab or F(ab′)2 blocks ligand-mediated internalization of CD22. In conclusion, these studies demonstrate that internalization of CD22 is dependent on its association with the AP-2 complex via tyrosine-based internalization motifs.