Mediated uptake of amino acids by membrane vesicles isolated from Balb/c 3T3 cells transformed by simian virus 40 has been demonstrated. Initial rates of transport of radioactively labeled L-leucine and alpha-aminoisobutyric acid were enhanced by the addition of NaCl (100 mM) to the reaction mixture at the start of the uptake process. This enhancement included a prominent "overshoot" during initial uptake. Slight stimulation of alpha-aminoisobutyric acid uptake was seen with K+, but none with Li+. The mediated nature of the uptake event for L-leucine was shown by saturation kinetics and by inhibition with L-valine. The transport assay measured predominantly intravesicular amino acid uptake rather than binding, as shown by the variation of uptake in response to changes in extravesicular osmolarity. Electron microscopy confirmed the presence of closed vesicles. Thus, amino acid transport has been characterized in an in vitro membrane vesicles system which should prove useful for studies of growth control.