Moderate hemolytic anemia, abnormal erythrocyte morphology (spherocytosis), and decreased membrane stability are observed in mice with complete deficiency of all erythroid protein 4.1 protein isoforms (4.1−/−; Shi TS et al. J Clin Invest 103: 331, 1999). We have examined the effects of erythroid protein 4.1 (4.1R) deficiency on erythrocyte cation transport and volume regulation. 4.1−/− mice exhibited erythrocyte dehydration that was associated with reduced cellular K and increased Na content. Increased Na permeability was observed in these mice, mostly mediated by Na/H exchange with normal Na-K pump and Na-K-2Cl cotransport activities. The Na/H exchange of 4.1−/− erythrocytes was markedly activated by exposure to hypertonic conditions (18.2 ± 3.2 in 4.1−/− vs. 9.8 ± 1.3 mmol/1013 cell × h in control mice), with an abnormal dependence on osmolality (EC50 = 417 ± 42 in 4.1−/− vs. 460 ± 35 mosmol/kgH2O in control mice), suggestive of an upregulated functional state. While the affinity for internal protons was not altered (K0.5 = 489.7 ± 0.7 vs. 537.0 ± 0.56 nM in control mice), the Vmax of the H-induced Na/H exchange activity was markedly elevated in 4.1−/− erythrocytes ( Vmax 91.47 ± 7.2 compared with 46.52 ± 5.4 mmol/1013 cell × h in control mice). Na/H exchange activation by okadaic acid was absent in 4.1−/− erythrocytes. Altogether, these results suggest that erythroid protein 4.1 plays a major role in volume regulation and physiologically downregulates Na/H exchange in mouse erythrocytes. Upregulation of the Na/H exchange is an important contributor to the elevated cell Na content of 4.1−/− erythrocytes.