Members of the P 4 family of P-type ATPases (P 4 -ATPases) are believed to function as phospholipid flippases in complex with CDC50 proteins. Mutations in the human class 1 P 4 -ATPase gene ATP8B1 cause a severe syndrome characterized by impaired bile flow (intrahepatic cholestasis), often leading to end-stage liver failure in childhood. In this study, we determined the specificity of human class 1 P 4 -ATPase interactions with CDC50 proteins and the functional consequences of these interactions on protein abundance and localization of both protein classes. ATP8B1 and ATP8B2 co-immunoprecipitated with CDC50A and CDC50B, whereas ATP8B4, ATP8A1, and ATP8A2 associated only with CDC50A. ATP8B1 shifted from the endoplasmic reticulum (ER) to the plasma membrane upon coexpression of CDC50A or CDC50B. ATP8A1 and ATP8A2 translocated from the ER to the Golgi complex and plasma membrane upon coexpression of CDC50A, but not CDC50B. ATP8B2 and ATP8B4 already displayed partial plasma membrane localization in the absence of CDC50 coexpression but displayed a large increase in plasma membrane abundance upon coexpression of CDC50A. ATP8B3 did not bind CDC50A and CDC50B and was invariably present in the ER. Our data show that interactions between CDC50 proteins and class 1 P 4 -ATPases are essential for ER exit and stability of both subunits. Furthermore, the subcellular localization of the complex is determined by the P 4 -ATPase, not the CDC50 protein. The interactions of CDC50A and CDC50B with multiple members of the human P 4 -ATPase family suggest that these proteins perform broader functions in human physiology than thus far assumed.