The recent characterization of a vesicular microvillous plasma membrane preparation from human placenta has made possible study of the characteristics of amino acid transport by the isolated membrane. Variation of medium osmolality demonstrated that alpha-aminoisobutyric acid (AIB) is taken up virtually entirely into a vesicular space. Vesicular volume was determined from equilibrium uptake of three dissimilar nonamino acid substrates, all of which gave a common value. Temperature dependence, saturability, specificity, and similarity of kinetic constants all indicated that AIB uptake by the vesicles resembles that by whole villous tissue. AIB uptake was stimulated specifically by an inward sodium gradient and unaffected by an outward potassium gradient. The persistence of stimulation up to 2 h after the gradient was established could be correlated with the slow equilibration of sodium across the membrane. AIB exodus could be studied with minimal problems of substrate recapture by using preloaded membrane vesicles bound tightly to cellulose ester filters to facilitate transfer to fresh medium. The exodus process was saturable with a Km considerably greater than that for entry.