The sodium-D-glucose cotransporter (SGLT1) was expressed in a yeast mutant strain NY 17 (sec6-4) that accumulates secretory vesicles at a nonpermissive temperature because of a block in the delivery of these vesicles to the plasma membrane. By differential centrifugation a microsomal fraction enriched in secretory vesicles was prepared with a high specific activity of the vanadate-sensitive H+-ATPase and invertase. In this membrane fraction one protein band of an apparent molecular weight of 55 kDa representing the nonglycosylated SGLT1 protein could be detected by immunochemical analysis. In addition, higher molecular weight protein bands probably representing dimers and aggregates were found. In transport studies with the microsomes D-glucose fluxes showed asymmetric properties: efflux experiments revealed the typical properties of the SGLT1 such as sodium dependence, inhibition by phlorizin and potential dependence. Influx of D-glucose showed no dependence on sodium and was not inhibited by phlorizin. Furthermore, the transporter exhibited a striking asymmetry with regard to the D-glucose affinity and the sugar specificity. These results suggest that the orientation of the SGLT1 expressed in yeast secretory vesicles is, indeed, inverted with regard to its configuration in the plasma membrane of epithelial cells. Moreover, there are striking functional differences between the periplasmic and cytoplasmic face of the transporter.