The adaptation of the capacity of the intestinal peptide transporter PEPT1 to varying substrate concentrations may be important with respect to its role in providing bulk quantities of amino acids for growth, development and other nutritional needs. We here describe a novel phenomenon of regulation of PEPT1 in the Xenopus oocyte system. Using electrophysiological and immunofluorescence methods we demonstrate, that a prolonged substrate exposure of rabbit PEPT1 (rPEPT1) causes a retrieval of transporters from the membrane. Capacitance as a measure of membrane surface area increased in parallel with the increase in rPEPT1-mediated transport currents with a slope of about 5% of basal surface per 100 nA. Exposure of oocytes to the model peptide Gly-L-Gln for 2 hrs resulted in a decrease in maximal transport currents with no change of membrane capacitance. However, exposure to substrate for 5 hrs decreased transport currents but in parallel also surface area by an endocytotic removal of transporter proteins from the surface. The reduction of the surface expression of rPEPT1 was confirmed by presteady-state current measurements and immunofluorescent labeling of rPEPT1. A similar simultaneous decrease of current and surface area was also observed, when endocytosis was stimulated by the activation of protein kinase C (PKC). Cytochalasin D inhibited all changes evoked by either to dipeptide or PKC-stimulation whereas the PKC-selective inhibitor bisindolylmaleimide only affected PKC-stimulated endocytotic processes but not substrate-dependent retrieval of rPEPT1. Co-expression studies with the human glucose transporter hSGLT1 revealed that substrate exposure affected selectively the peptide transporter, but not activity of hSGLT1.