We recently reported that a considerable amount of the sodium D-glucose cotransporter (SGLT1) present in Caco-2 cells, a model for human enterocytes, is located in intracellular compartments, which were attached to microtubules (Kipp et al., Am. J. Physiol. 285: C737, 2003). A similar distribution pattern was also observed in enterocytes in thin sections from human jejunum highlighting the validity of the Caco-2 cell model. Fluorescent surface labeling of live Caco-2 cells revealed that the intracellular compartments containing SGLT1 were accessible by endocytosis. To elucidate the role of endosomal SGLT1 in the regulation of sodium-dependent D-glucose uptake into enterocytes, we compared SGLT1 mediated D-glucose uptake into Caco-2 cells with the subcellular distribution of SGLT1 after challenging the cells with different stimuli. Incubation (90 min) of Caco-2 cells with mastoparan (50 µM), a drug which enhances apical endocytosis, shifted a large amount of SGLT1 from the apical membrane to intracellular sites and significantly reduced (60%) sodium-dependent -[¹⁴C]methyl-D-glucose uptake. We also investigated the effect of altered extracellular D-glucose levels. Cells pre-incubated (1 h) with D-glucose free medium exhibited a significantly higher sodium-dependent -[¹⁴C]methyl-D-glucose uptake (45%) than cells pre-incubated with high D-glucose medium (100 mM, 1 h). Interestingly, regulation of SGLT1-mediated D-glucose uptake into Caco-2 cells by extracellular D-glucose levels occurred without redistribution of cellular SGLT1. These data suggest that pharmacologically D-glucose uptake can be regulated by a shift of SGLT1 between the plasma membrane and the endosomal pool, however, regulation by the physiological substrate D-glucose can only be explained by an alternative mechanism.