5-Thio-D-glucose ( 5TG ) elicits a time-dependent effect on net D-glucose transport and metabolism in the mouse small intestine. When incubation periods were less than 45 min, 5TG inhibited net D-glucose transport. As incubation periods were lengthened to 60 min or greater, net D-glucose transport was potentiated by 5TG , with a concomitant inhibition of lactate production from exogenous D-glucose. Furthermore, D-glucose inhibited 5TG transport at every time point studied. Initial velocity profiles done with 5TG and D-glucose, respectively, resulted in a KT of 6.3 mM and a Tmax of 18.5 mumole/g/30 min for 5TG ; while for D-glucose the KT was 11.7 mM and the Tmax was 43.4 mumole/g/30 min. Competitive inhibition between 5TG and D-glucose was demonstrated. The apparent KI's for 5TG and D-glucose were 3.0 mM and 9.3 mM, respectively. These findings indicated that 5TG has a lower affinity for the hexose-transport carrier than D-glucose. 5TG was also found to inhibit 3-O-methyl-D-glucose (3MG) and D-galactose transport in a dose-dependent manner. Basal levels of lactate production were not affected by 5TG nor did 5TG affect lactate levels in the presence of 3MG. These results suggest that 5TG decreases intestinal utilization of D-glucose via glycolysis to partially account for the observed increase in net transmural transport of D-glucose.