A facilitated diffusion for glycerol is present in human erythrocytes. Glycerol transporters identified to date belong to the Major Intrinsic Protein (MIP) family of integral membrane proteins, and one of them, aquaporin-3 (AQP3), has been characterized in mammals. Using an antibody raised against a peptide corresponding to the rat AQP3 carboxyl terminus, we examined the presence of AQP3 in normal and Colton-null (aquaporin-1 (AQP1)-deficient) human erythrocytes. Three immunoreactive bands were detected on immunoblots of both normal and Colton-null red cells, very similar to the bands revealed in rat kidney, a material in which AQP3 has been extensively studied. By immunofluorescence, anti-AQP3 antibodies stained the plasma membranes of both normal and Colton-null erythrocytes. Glycerol transport was measured on intact erythrocytes by stopped-flow light scattering and on one-step pink ghosts by a rapid filtration technique. Glycerol permeability values, similar in both cell types, suggest that AQP1 does not represent the major path for glycerol movement across red blood cell membranes. Furthermore, pharmacological studies showed that Colton-null red cells remain sensitive to water and glycerol flux inhibitors, supporting the idea that another proteinaceous path, probably AQP3, mediates most of the glycerol movements across red cell membranes and represents part of the residual water transport activity found in AQP1-deficient red cells.