The initial stages in the biosynthesis of the cloned Na+/glucose cotransporter were examined by in vitro expression of the protein in the absence and presence of pancreatic microsomes. Glycosylation was detected by endoglycosidase-H shifts in the apparent size of the proteins on SDS-PAGE. In the presence of microsomes, Mr increased from 52,000 to 58,000, and this was reversed by endo-H. This demonstrates that the protein is glycosylated and that there is no large cleavable signal sequence. Using partial transcripts and site-directed mutagenesis, we established that Asn-248 is glycosylated. Glycosylation was not required for the functional expression of the transporter in Xenopus oocytes. In terms of the topology of the protein, these results suggest that Asn-248 is on the external surface of the membrane.