The aquaporin-1 (AQP1) water transport protein contains a polymorphism corresponding to the Colton red blood cell antigens. To define the fraction of membrane water permeability mediated by AQP1, red cells were obtained from human kindreds with the rare Colton-null phenotype. Homozygosity or heterozygosity for deletion of exon I in AQP1 correlated with total or partial deficiency of AQP1 protein. Homozygote red cell morphology appeared normal, but clinical laboratory studies revealed slightly reduced red cell life span in vivo; deformability studies revealed a slight reduction in membrane surface area. Diffusional water permeability (Pd) was measured under isotonic conditions by pulsed field gradient NMR. Osmotic water permeability (Pf) was measured by change in light scattering after rapid exposure of red cells to increased extracellular osmolality. AQP1 contributes approximately 64% (Pd = 1.5 x 10(-3) cm/s) of the total diffusional water permeability pathway, and lipid permeation apparently comprises approximately 23%. In contrast, AQP1 contributes > 85% (Pf = 19 x 10(-3) cm/s) of the total osmotic water permeability pathway, and lipid permeation apparently comprises only approximately 10%. The ratio of AQP1-mediated Pf to Pd predicts the length of the aqueous pore to be 36 A.