The previously undescribed heterozygous missense mutation E758K was discovered in the human AE1/SLC4A1/band 3 gene in two unrelated patients with well-compensated hereditary spherostomatocytic anemia (HSt). Oocyte surface expression of AE1 E758K, in contrast to that of wild-type AE1, required coexpressed glycophorin A (GPA). The mutant polypeptide exhibited, in parallel, strong GPA dependence of DIDS-sensitive 36Cl− influx, trans-anion-dependent 36Cl− efflux, and Cl−/HCO3− exchange activities at near wild-type levels. AE1 E758K expression was also associated with GPA-dependent increases of DIDS-sensitive pH-independent SO42− uptake and oxalate uptake with altered pH dependence. In marked contrast, the bumetanide- and ouabain-insensitive 86Rb+ influx associated with AE1 E758K expression was largely GPA-independent in Xenopus oocytes and completely GPA-independent in Ambystoma oocytes. AE1 E758K-associated currents in Xenopus oocytes also exhibited little or no GPA dependence. 86Rb+ influx was higher but inward cation current was lower in oocytes expressing AE1 E758K than previously reported in oocytes expressing the AE1 HSt mutants S731P and H734R. The pharmacological inhibition profile of AE1 E758K-associated 36Cl− influx differed from that of AE1 E758K-associated 86Rb+ influx, as well as from that of wild-type AE1-mediated Cl− transport. Thus AE1 E758K-expressing oocytes displayed GPA-dependent surface polypeptide expression and anion transport, accompanied by substantially GPA-independent, pharmacologically distinct Rb+ flux and by small, GPA-independent currents. The data strongly suggest that most of the increased cation transport associated with the novel HSt mutant AE1 E758K reflects activation of endogenous oocyte cation permeability pathways, rather than cation translocation through the mutant polypeptide.