I t has been proposed that the mercurial-sensitive water transporter in mammalian erythrocytes is the anion exchanger band 3 (AE1) and/or the glucose transporter, band 4.5 (GLUT1). Using a functional assay for water channel expression in Xenopus oocytes (Zhang, R., K. A. Logee, and A. S. Verkman. 1990. J. Biol. Chem. 265:15375-15378), we compared osmotic water permeability (Pf) of oocytes injected with water, reticulocyte mRNA, AE1 mRNA, and GLUT1 mRNA. Injection of oocytes with 5-50 ng of in vitro-transcribed AE1 mRNA had no effect on Pf, but increased trans-stimulated 36Cl uptake greater than fourfold in a dinitro-disulfonic stilbene (DNDS)-inhibitable manner. Injection with 1-50 ng of in vitro-transcribed GLUT1 mRNA increased 3H-methylglucose uptake greater than 15-fold in a cytochalasin B-sensitive manner and increased Pf from (3.7 +/- 0.4) x 10(-4) cm/s (SE, n = 16, 10 degrees C) in water-injected oocytes up to (13 +/- 1) x 10(-4) cm/s (n = 18). Both the increments in sugar and water transport were inhibited by cytochalasin B (25 microM) and phloretin (0.2 mM); neither was inhibited by 0.3 mM HgCl2. In oocytes injected with 50 ng of rabbit reticulocyte mRNA, the Pf of (18 +/- 2) x 10(-4) cm/s (n = 18) was reduced to (4.0 +/- 0.6) x 10(-4) cm/s (n = 10) by HgCl2, but was not inhibited by DNDS (0.4 mM), cytochalasin B or phloretin. Coinjection of reticulocyte mRNA with antisense oligodeoxyribonucleotides against AE1 or GLUT1 did not affect Pf, but inhibited completely the incremental uptake of 36Cl or 3H-methylglucose, respectively. Expression of size-fractionated mRNA from reticulocyte gave a 2-2.5-kb size for water channel mRNA, less than the 4-4.5-kb size for the Cl transporter. These results provide evidence that facilitated water transport in erythrocytes is mediated not by bands 3 or 4.5, but by distinct water transport protein(s).