The membrane topology of the high affinity, Na(+)-coupled L-glutamate/L-aspartate transporter (GLAST-1) of the central nervous system has been determined. Truncated GLAST-1 cDNA constructs encoding protein fragments with an increasing number of hydrophobic regions were fused to a cDNA encoding a reporter peptide with two N-glycosylation sites. The respective cRNA chimeras were translated in vitro and in vivo in Xenopus oocytes. Posttranslational N-glycosylation of the two reporter consensus sites monitors the number, size, and orientation of membrane-spanning domains. The results of our experiments suggest a novel 10-transmembrane domain topology of GLAST-1, a representative of the L-glutamate neurotransmitter transporter family, with its NH2 and COOH termini on the cytoplasmic side, six NH2-terminal hydrophobic transmembrane alpha-helices, and four COOH-terminal short hydrophobic domains spanning the bilayer predicted as beta-sheets.