Transport through the microvillous membrane of the syncytium is the first step in placental transfer of nutrients. We have therefore studied glucose transport by isolated microvillous membrane vesicles. Transport occurred by selective and rapid facilitated diffusion, which was inhibited by phloridzin, phloretin, cytochalasin B, and HgCl2. Nonmediated diffusion of the substrate was found by three independent methods to be very slow. Competition studies showed that aldohexoses in the C-1 chair conformation were the preferred substrates. Transport was independent of sodium gradients and was not modulated by insulin. However, several steroids inhibited transport including estriol and progesterone, which are abundant in utero. Kinetic analysis by equilibrium exchange demonstrated a Km of 31 mM and a Vmax of 120 nmol . s-1 . mg protein-1. The Km and Vmax suggest a large capacity in relation to calculated fetal needs. In consequence of this capacity, intrasyncytial concentrations of glucose are probably maintained near those of maternal blood. Augmentation of transport at this membrane by hormones or other agents is unlikely to increase fetal supply of glucose, but down regulation by steroids may serve a regulatory function.