Continuous labeling of primary cultures of human syncytiotrophoblasts with [125I]iodoepidermal growth factor (iodo-EGF) at 37 C revealed a progressive increase in intracellular EGF with 5% of total label observed in intracellular organelles at 2 min and 88% by 60 min. Detailed analysis by electron microscope radioautography showed a progressive transfer of [125I]iodo-EGF from microvillar processes to the bases of microvilli followed by uptake into vesicular and polymorphic endosomes as well as multivesicular bodies. Quantitative analysis revealed an association of radiolabel with the membrane of vesicular endosomes but more generally dispersed over the content of multivesicular bodies. Chloroquine (100 microM), colchicine (100 microM), and bacitracin (1 mg/ml) augmented EGF binding from 45% specific binding in control cultures to 60%, 60%, and 65% specific binding, respectively, with a majority of the ligand (72-83%) observed in vesicular and polymorphic endosomes as well as in multivesicular bodies, i.e. similar to controls. Functional studies indicated a significant enhancement of [14C]-3-O-methylglucose uptake by EGF. Chloroquine-treated cell cultures significantly augmented the stimulation of 3-O-methylglucose uptake by EGF.