1. Rat small intestinal basolateral membrane vesicles (BLMVs) were prepared and found to be 31% non-vesiculated and 69% vesiculated, 4.9% right side out and 63.8% inside out. 2. Thiamine uptake by BLMVs followed a hyperbolic time course reaching equilibrium after 60-90 min incubation. Uptake was not affected by the transmembrane potential or by the presence or absence of Na+ or K+ in the incubation medium. 3. At concentrations below 1.25 microM, [3H]thiamine was taken up mainly by a saturable mechanism with an apparent Michaelis-Menten constant (Km) = 1.32 microM and maximal flux (Jmax) = 1.93 pmol (mg protein)-1 (4 s)-1. At higher concentrations, a non-saturable mechanism prevailed. 4. Only 29% of [3H]thiamine taken up by the vesicles was membrane bound, the remaining being translocated into the vesicular space. No thiamine phosphoesters could be detected inside the vesicles. 5. In the absence of ATP, the Na(+)-K(+)-ATPase inhibitors ouabain, frusemide and vanadate reduced thiamine uptake by 35, 30 and 15% respectively. 6. In experiments conducted with K+ inside the vesicles and Na+, Mg2+ and ATP outside, the time course of thiamine uptake by BLMVs displayed an overshoot (80-90% increment) at 30 s incubation as compared to controls. When ATP was replaced with phosphocreatine, or when NaCl was replaced with isosmotic amounts of KCl, the overshoot disappeared. 7. The thiamine analogues pyrithiamine, amprolium and 4'-oxythiamine decreased the ATPase-dependent transport of [3H]thiamine by 100, 86 and 31% respectively. 8. These results provide evidence that the transport of thiamine by BLMVs is coupled directly to the hydrolysis of ATP (primary active transport).