Sodium transport into human placental brush border membrane vesicles was examined in the presence of an outwardly directed sodium gradient leading to the formation of an intravesicular negative charge. 22Na entered the vesicles in a time dependent fashion. The activation energy of the uptake process was calculated and was found to be 11.2 kcal/mol, similar to the value of ionic diffusion in free solution. Amiloride inhibited Na uptake in a concentration dependent fashion with an IC50 value of 3.08 microM. Neither ouabain nor bumetanide had an effect on Na uptake at concentrations up to 100 or 1000 microM, respectively. The system presented here indicates Na transport via channels without involvement of the Na-K-ATPase or the Na-K-Cl cotransporter. The system may be useful in investigating Na transport defects in cystic fibrosis.