1. The regulation of glucose transport by physiological concentrations of insulin was investigated using a preparation of rat jejunum perfused in situ with 5 mM glucose on both sides. 2. Luminal uptake was 87% inhibited (P < 0.001) by 0.2 mM phlorizin, indicating that it occurred by means of the Na(+)-D-glucose cotransporter. Vascular uptake was completely abolished by 0.2 mM phloretin, indicating that it was facilitated in nature. 3. When infused into the vascular circuit, insulin (10(-11) to 10(-7) M) stimulated vascular, and inhibited luminal, glucose uptake to a similar extent. Maximal stimulation of vascular uptake was increased by 40% compared with control infusions (P < 0.01) and occurred at 10(-10) M insulin. These effects were independent of changes in metabolism and vascular glucose concentration. 4. The time taken for half-maximal stimulation of vascular uptake was 6.3 +/- 0.7 min and preceded that for inhibition of luminal uptake by 6.5 +/- 1.3 min (P < 0.02). 5. The rapid inhibition of luminal glucose uptake by the acute administration of insulin was also detected by perfusion of jejunal loops in vivo. 6. It is concluded that the transport steps involved in intestinal glucose uptake are subject to rapid regulation by physiological concentrations of insulin and that the initial site of action is on the vascular side.