The change of the acridine orange absorbance was used to monitor the formation and/or dissipation of a pH gradient in microvillous membrane vesicles (MVV) isolated from human term placenta. Under Na⁺ efflux conditions, an acidification of the intravesicular space occured and it was completely inhibited by 0.1 mM amiloride. Under K⁺ efflux conditions, an acidification of the intravesicular space occured and it was potentiated by valinomycin or FCCP. An inwardly directed chloride gradient also induced a minor intravesicular acidification, but it was not observed in voltage-clampled MVV. The initial rate of the dissipation of a pH gradient was accelerated by pulse injections of Na⁺ in a saturable manner and Li⁺ could replace Na⁺. The kinetic parameter of Na⁺ in placental Na⁺/H⁺ exchange was similar to that of renal Na⁺/H⁺ exchange. Amiloride was a inhibitor of directly coupled Na⁺/H⁺ exchange and its IC₅₀ in placental MVV was about 14-fold higher than that in renal brush border membrane. These results indicate that Na⁺/H⁺ exchanger exists in human placental MVV and that its kinetic characteristics is similar to that of renal Na⁺/H⁺ exchanger but its pharmacological characteristics is different. In placental MVV K⁺, H⁺, and, relatively minor chloride conductances are present. The magnitude of Cl^-/OH^- exchange, even though it exists, seems to be smaller than that of Na⁺/H⁺ exchange.