Protein 4.2 is a major component of the red blood cell (RBC) membrane skeleton. We used targeted mutagenesis in embryonic stem (ES) cells to elucidate protein 4.2 functions in vivo. Protein 4.2–null (4.2^–/–) mice have mild hereditary spherocytosis (HS). Scanning electron microscopy and ektacytometry confirm loss of membrane surface in 4.2^–/– RBCs. The membrane skeleton architecture is intact, and the spectrin and ankyrin content of 4.2^–/– RBCs are normal. Band 3 and band 3–mediated anion transport are decreased. Protein 4.2^–/– RBCs show altered cation content (increased K⁺/decreased Na⁺)resulting in dehydration. The passive Na⁺ permeability and the activities of the Na-K-2Cl and K-Cl cotransporters, the Na/H exchanger, and the Gardos channel in 4.2^–/– RBCs are significantly increased. Protein 4.2^–/– RBCs demonstrate an abnormal regulation of cation transport by cell volume. Cell shrinkage induces a greater activation of Na/H exchange and Na-K-2Cl cotransport in 4.2^–/– RBCs compared with controls. The increased passive Na⁺ permeability of 4.2^–/– RBCs is also dependent on cell shrinkage. We conclude that protein 4.2 is important in the maintenance of normal surface area in RBCs and for normal RBC cation transport.