The effect of zinc on sodium coupled glucose uptake was studied in pig intestinal brush border membrane vesicles. In this system zinc inhibited glucose uptake and appeared to have a Ki of 0.25 mM. When tested by spectrophotometry, electron microscopy and protein determination following centrifugation, no evidence of significant vesicle aggregation was found with 0.5 mM zinc treatment. Zinc inhibition of glucose uptake persisted when the vesicle membrane potential was clamped with identical KCl concentrations inside and outside the vesicles in the presence of valinomycin. Variation of the glucose and sodium concentrations gave results indicating that zinc reduces glucose affinity for the carrier but not sodium binding to the transporter. The glucose inhibitory effect was not due to a rapid dissipation of the sodium gradient as zinc failed to affect sodium uptake in the absence of glucose. Zinc also failed to inhibit glucose efflux from vesicles under isotopic exchange conditions, when glucose and sodium concentrations were identical inside and outside vesicles. The t1/2 of glucose inhibition by zinc was relatively long, i.e. 6 min. We conclude that zinc acts as an inhibitor of glucose transport by interacting with the sodium-glucose co-transporter. The long zinc incubation time required to achieve maximal inhibition of glucose transport suggests that this interaction takes place within vesicles.