In the present work we studied the effects of phloretin on Na+-dependent D-glucose uptake by brush border membrane vesicles isolated from rabbit small intestine. Phloretin had no inhibitory effect on Na+-dependent D-glucose uptake in the presence of equilibrated Na+, although it inhibited the process in the presence of a Na+ gradient. Phlorizin inhibited Na+-dependent D-glucose uptake both in the presence and in the absence of a Na+ gradient. Phloretin and phlorizin competed with each other for the inhibition of Na+-energized D-glucose uptake. These results indicate that phloretin has no direct interaction with the Na+-dependent D-glucose transporter per se, though phloretin and phlorizin may have a common step(s) in their inhibition mechanisms. Phloretin, but not phlorizin, was found to increase the equilibrium level of D-glucose uptake whether Na+ was present or not. The increase in the equilibrium level was due to phloretin-induced binding of D-glucose to the brush border membrane, but not due to an increase in the intravesicular space. It seems to occur by a mechanism different from that by which phloretin inhibits Na+-energized D-glucose uptake, because the equilibrium level of D-glucose uptake is increased by the ionized form of phloretin but Na+-energized D-glucose uptake is inhibited by the unionized form.