The Tweety proteins comprise a family of chloride ion channels with three members identified in humans (TTYH1-3) and orthologues in fly and murine species. In humans, increased TTYH2 expression is associated with cancer progression, whereas fly Tweety is associated with developmental processes. Structurally, Tweety proteins are characterized by five membrane-spanning domains and N-glycan modifications important for trafficking to the plasma membrane, where these proteins are oriented with the amino terminus located extracellularly and the carboxyl terminus cytoplasmically. In addition to N-glycosylation, ubiquitination mediated by the HECT type E3 ubiquitin ligase Nedd4-2 is a post-translation modification important in regulating membrane proteins. In the present study, we performed a comprehensive analysis of the ability of each of TTYH1-3 to interact with Nedd4-2 and to be ubiquitinated and regulated by this ligase. Our data indicate that Nedd4-2 binds to two family members, TTYH2 and TTYH3, which contain consensus PY ((L/P)PXY) binding sites for HECT type E3 ubiquitin ligases, but not to TTYH1, which lacks this motif. Consistently, Nedd4-2 ubiquitinates both TTYH2 and TTYH3. Importantly, we have shown that endogenous TTYH2 and Nedd4-2 are binding partners and demonstrated that the TTYH2 PY motif is essential for these interactions. We have also shown that Nedd4-2-mediated ubiquitination of TTYH2 is a critical regulator of cell surface and total cellular levels of this protein. These data, indicating that Nedd4-2 differentially interacts with and regulates TTYH1-3, will be important for understanding mechanisms controlling Tweety proteins in physiology and disease.