The mechanism for L-lactate transport across microvillous membrane vesicles prepared from rat placenta was examined. Uptake of L-lactate into these vesicles was mainly the result of transport into the intravesicular (osmotically active) space. The initial rate of L-lactate uptake was not affected by the presence of an inward gradient of either Na+ or K+. In the presence of an inward-directed proton gradient, L-lactate uptake was markedly stimulated, accumulating at concentrations 6-7-fold higher than the equilibrium. Lower transmembrane pH gradients were associated with slower initial uptakes and smaller overshoots. L-Lactate uptake determined under an inside-directed pH gradient was strongly inhibited by p-chloromercuriphenylsulphonic acid, a protein-thiol oxidizing agent. L-Lactate uptake was: (1) saturable as a function of the concentration of L-lactate, (2) inhibited by monocarboxylic acids such as pyruvate, D-lactate, beta-hydroxybutyrate and alpha-cyano-4-hydroxycinnamic acid, and (3) temperature-dependent. When present inside the vesicles, L-lactate, pyruvate and beta-hydroxybutyrate caused trans-stimulation of L-lactate uptake both in the presence and in the absence of an inside-directed pH gradient, indicating that L-lactate transport is a reversible process that can be shared by other monocarboxylic acids. There were no significant changes in maximal initial rate or in the kinetic parameters of L-lactate transport during the last 3 days of gestation.