Drugs are transported by cotransporters with widely different turnover rates. We have examined the underlying mechanism using, as a model system, glucose and indican (indoxyl-beta-D-glucopyranoside) transport by human Na+/glucose cotransporter (hSGLT1). Indican is transported by hSGLT1 at 10% of the rate for glucose but with a fivefold higher apparent affinity. We expressed wild-type hSGLT1 and mutant G507C in Xenopus oocytes and used electrical and optical methods to measure the kinetics of glucose (using nonmetabolized glucose analogue alpha-methyl-D-glucopyranoside, alphaMDG) and indican transport, alone and together. Indican behaved as a competitive inhibitor of alphaMDG transport. To examine protein conformations, we recorded SGLT1 capacitive currents (charge movements) and fluorescence changes in response to step jumps in membrane voltage, in the presence and absence of indican and/or alphaMDG. In the absence of sugar, voltage jumps elicited capacitive SGLT currents that decayed to steady state with time constants (tau) of 3-20 ms. These transient currents were abolished in saturating alphaMDG but only slightly reduced (10%) in saturating indican. SGLT1 G507C rhodamine fluorescence intensity increased with depolarizing and decreased with hyperpolarizing voltages. Maximal fluorescence increased approximately 150% in saturating indican but decreased approximately 50% in saturating alphaMDG. Modeling indicated that the rate-limiting step for indican transport is sugar translocation, whereas for alphaMDG it is dissociation of Na+ from the internal binding sites. The inhibitory effects of indican on alphaMDG transport are due to its higher affinity and a 100-fold lower translocation rate. Our results indicate that competition between substrates and drugs should be taken into consideration when targeting transporters as drug delivery systems.