K -Cl cotransport activity in rbc is a major determinant of rbc volume and density. Pathologic activation of erythroid K-Cl cotransport activity in sickle cell disease contributes to rbc dehydration and cell sickling. To address the roles of individual K-Cl cotransporter isoforms in rbc volume homeostasis, we disrupted the Kcc1 and Kcc3 genes in mice. As rbc K-Cl cotransport activity was undiminished in Kcc1^–/– mice, decreased in Kcc3^–/– mice, and almost completely abolished in mice lacking both isoforms, we conclude that K-Cl cotransport activity of mouse rbc is mediated largely by KCC3. Whereas rbc of either Kcc1^–/– or Kcc3^–/– mice were of normal density, rbc of Kcc1^–/–Kcc3^–/– mice exhibited defective volume regulation, including increased mean corpuscular volume, decreased density, and increased susceptibility to osmotic lysis. K-Cl cotransport activity was increased in rbc of SAD mice, which are transgenic for a hypersickling human hemoglobin S variant. Kcc1^–/–Kcc3^–/– SAD rbc lacked nearly all K-Cl cotransport activity and exhibited normalized values of mean corpuscular volume, corpuscular hemoglobin concentration mean, and K⁺ content. Although disruption of K-Cl cotransport rescued the dehydration phenotype of most SAD rbc, the proportion of the densest red blood cell population remained unaffected.