The uptake of L-lysine in human placental microvilli vesicles prepared from human term placenta was studied using the rapid filtration technique. The uptake of L-lysine into the vesicles was osmotically sensitive. This finding indicates that the uptake of L-lysine by placental microvilli vesicles represents transport into the membrane vesicles. The uptake of L-lysine into microvilli vesicles is sodium independent. The initial rate of uptake is markedly increased when the intravesicular space is rendered electrically more negative by membrane diffusion potential induced by the use of highly permeant anions or by K+ diffusion potentials via valinomycin. These results indicate that the sodium independent uptake of L-lysine into the microvilli membrane vesicles is dependent on the electrical potential difference of the membranes. A kinetic analysis of the uptake demonstrated that two transport systems for vesicular entry of L-lysine existed with a Km1 of 0.13 mM, Vmax1 of 590 pmol/mg protein/20 sec, Km2 of 0.91 mM, Vmax2 of 2010 pmol/mg protein/20 sec. The uptake of L-lysine in microvilli vesicles was inhibited by dibasic amino acid (L-arginine, L-ornithine, L-glutamine), but not by other classes of amino acid. These results indicate the existence of a dibasic amino acid specific transport system in placental microvilli membrane.