We have examined the expression and cellular location of facilitated glucose transporter proteins (GLUT1, -3, and -5) in a human colonic epithelial cell line (Caco-2) by using peptide-specific antibodies. A differential cellular distribution of these transporters was observed in differentiated (greater than 14 days postconfluence) Caco-2 cells by immunofluorescence and immunoelectron microscopy. GLUT1 was localized primarily to the basolateral membrane, whereas GLUT3 was predominantly localized to the apical membrane. GLUT5, which was detected in only approximately 40% of fully differentiated Caco-2 cells, was found primarily in the apical membrane but was also present in both basolateral and intracellular membranes. A Na(+)-independent glucose transport system in the apical membrane of Caco-2 cells has been described previously [Blais, A., Bissonnette, A. & Berteloot, A. (1987) J. Membr. Biol. 99, 113-125], and we propose that GLUT3 mediates this process. The amino acid sequence identity (57%) and structural conservation between GLUT1 and GLUT3 may make these transporters an ideal model system for examining the molecular basis for polarized sorting of membrane proteins.