The actin motor myosin VI regulates endocytosis of cystic fibrosis transmembrane conductance regulator (CFTR) in the intestine, but the endocytic adaptor linking CFTR to myosin VI is unknown. Dab2 (Disabled 2) is the binding partner for myosin VI, clathrin, and alpha-AP-2 and directs endocytosis of low density lipoprotein receptor family members by recognizing a phosphotyrosine-binding domain. However, CFTR does not possess a phosphotyrosine-binding domain. We examined whether alpha-AP-2 and/or Dab2 were binding partners for CFTR and the role of myosin VI in localizing endocytic adaptors in the intestine. CFTR co-localized with alpha-AP-2, Dab2, and myosin VI and was identified in a complex with all three endocytic proteins in the intestine. Apical CFTR was increased in the intestines of Dab-2 KO mice, suggesting its involvement in regulating surface CFTR. Glutathione S-transferase pulldown assays revealed binding of CFTR to alpha-AP-2 (but not Dab2) in the intestine, whereas Dab-2 interacted with alpha-AP-2. siRNA silencing of alpha-AP-2 in cells significantly reduced CFTR endocytosis, further supporting alpha-AP-2 as the direct binding partner for CFTR. alpha-AP-2 and Dab2 localized to the terminal web regions of enterocytes, but Dab2 accumulated in this location in Snell's Waltzer myosin VI((sv/sv)) intestine. Ultrastructural examination revealed that the accumulation of Dab2 correlated with prominent involution and the loss of normal positioning of the intermicrovillar membranes that resulted in expansion of the terminal web region in myosin VI((sv/sv)) enterocytes. The findings support alpha-AP-2 in directing myosin VI-dependent endocytosis of CFTR and a requirement for myosin VI in membrane invagination and coated pit formation in enterocytes.