Rat glucose transporter isoform 1 or rGLUT1, which is expressed in neonatal heart and the epithelial cells that form the blood-brain barrier, facilitates uptake of the trivalent arsenicals arsenite as As(OH)₃ and methylarsenite as CH₃As(OH)₂. GLUT1 may be the major pathway for arsenic uptake into heart and brain, where the metalloid causes cardiotoxicity and neurotoxicity. In this paper, we compare the translocation properties of GLUT1 for trivalent methylarsenite and glucose. Substitution of Ser(66), Arg(126) and Thr(310), residues critical for glucose uptake, led to decreased uptake of glucose but increased uptake of CH₃As(OH)₂. The K(m) for uptake of CH₃As(OH)₂ of three identified mutants, S66F, R126K and T310I, were decreased 4-10 fold compared to native GLUT1. The osmotic water permeability coefficient (P(f)) of GLUT1 and the three clinical isolates increased in parallel with the rate of CH₃As(OH)₂ uptake. GLUT1 inhibitors Hg(II), cytochalasin B and forskolin reduced uptake of glucose but not CH₃As(OH)₂. These results indicate that CH₃As(OH)₂ and water use a common translocation pathway in GLUT1 that is different to that of glucose transport.