Cd2+ uptake by brush border membrane vesicles isolated from rat small intestine was investigated by a rapid filtration technique. Cd2+ uptake showed time- and temperature-dependence. Changes in medium osmolarity had no effect on Cd2+ uptake, indicating that the process involves primarily binding of Cd2+ to membrane component(s). Membrane-impermeable chelating agents removed only about 60-65% of the bound cadmium from intact vesicles preincubated with Cd; almost all Cd was removed by chelators when membrane permeability was increased by 1-butanol. Thus Cd uptake by membrane vesicles results in binding to both sides of the membrane. Pretreatment of vesicles with cadmium resulted in a time-dependent inhibition of D-glucose uptake in the presence of a sodium (but no potassium) gradient. This inhibition is correlated with Cd binding to external sites on the D-glucose transporter, and not the driving force for D-glucose transport.