Abstract Active amino acid transport in Staphylococcus aureus U-71 membrane vesicles is coupled to either α-glycerol phosphate dehydrogenase or l-lactate dehydrogenase depending upon the growth conditions of the parent cells. Vesicles prepared from cells grown on gluconate as a primary carbon source exhibit an absolute specificity for α-glycerol phosphate as a physiological electron donor for transport, whereas vesicles prepared from cells grown on glucose as a primary carbon source exhibit an absolute specificity for l-lactate as an electron donor for transport. Both preparations exhibit similar dehydrogenase activities qualitatively, indicating that the coupling between these dehydrogenases and transport is altered. l-Lactate oxidation, l-lactate:dichlorophenolindophenol reductase activity, and l-lactate-dependent amino acid transport exhibit similar apparent Michaelis constants with respect to l-lactate, indicating that l-lactate oxidation per se is the rate-limiting step for amino acid transport in the appropriate membrane preparation. Amino acid transport is dependent on electron transfer, and inhibition of l-lactate oxidation by anaerobiosis, cyanide, 2-heptyl-4-hydroxyquinoline-N-oxide, amytal, and oxalate is directly related to inhibition of amino acid transport. However, only anaerobiosis, cyanide, 2-heptyl-4-hydroxyquinoline-N-oxide, and amytal, each of which inhibits electron transfer after the site of energy coupling, cause efflux. Oxalate, a potent inhibitor of l-lactate dehydrogenase, does not cause efflux despite almost complete inhibition of l-lactate oxidation and amino acid transport. Moreover, oxalate blocks or inhibits efflux caused by each of the other inhibitors and by 2,4-dinitrophenol. These results provide further evidence that active transport is dependent on the oxidation-reduction potential of the respiratory chain at the site of energy coupling. Cyanide-induced efflux is a saturable process with an apparent affinity constant that is approximately 500 times higher than the affinity constant for active transport. The apparent maximum velocity of efflux, on the other hand, is the same as that of active transport. These findings suggest that one of the primary effects of energy coupling is to change the affinity of the carrier for substrate. Under anaerobic conditions serine uptake exhibits linear kinetics, indicating that the rate-limiting step for serine uptake under these conditions is a nonsaturable process with an infinite Km. Moreover, approximately 5 min is required for external serine to equilibrate with the intramembranal pool at a variety of concentrations. Thus, it is highly unlikely that facilitated diffusion is the rate-limiting step for active serine uptake.