The domains responsible for the fructose specificity of GLUT5 were investigated by creating chimeras of GLUT5 with the selective glucose transporter GLUT3, which were expressed in Xenopus oocytes. 3-O-Methylglucose uptake of chimeric GLUT3-5 (M11; GLUT3 to the 11th transmembrane domain, GLUT5 to the carboxyl end) was similar to that of GLUT3, while fructose was not transported. Fructose uptake of chimeric GLUT5-3 (M3-5) to -5 (GLUT3 from the 3rd to 5th transmembrane domains, the rest GLUT5) was similar to that of GLUT5; no glucose was transported. Four chimeras transported neither fructose nor glucose: GLUT3-5 (M5; GLUT3 to the 5th transmembrane domain, GLUT5 to the carboxyl end), GLUT5-3 (M2; GLUT5 to the 2nd transmembrane domain, the rest GLUT3), GLUT5-3 (M3-11) to -5 (GLUT3 between the 3rd and 11th transmembrane domains, the rest GLUT5) and GLUT5-3 (M3-5) to -5-3 (M11; GLUT3 from the 3rd to 5th transmembrane domains and after the 11th transmembrane domain, the rest GLUT5). They, nevertheless, induced full-size proteins that were transported to the cell surface, as demonstrated by exofacial labeling with biotin. To conclude, the GLUT5 domain from the amino-terminus to the third transmembrane domain and that between the 5th and 11th transmembrane stretches seem to be necessary for fructose uptake.