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 also 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 x h in control), with an abnormal dependence on osmolarity, (K_0.5 = 417 ± 42 in 4.1 -/- vs. 460 ± 35 mOsm in control) suggestive of an up-regulated functional state. While the affinity for internal protons was not altered (K_0.5 = 489.7 ± 0.7 vs. 537.0 ± 0.56 nM in control mice), the V_max of the H-induced Na/H exchange activity was markedly elevated in 4.1-/- (V_max91.47 ± 7.2 compared to 46.52 ± 5.4 mmol/10¹³ cell x 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 down-regulates Na/H exchange in mouse erythrocytes. Up-regulation of Na/H exchange is an important contributor to the elevated cell Na content of 4.1 -/- erythrocytes.