Human glandular endometrial epithelial cells were cultured on porous tissue culture inserts to form tight, confluent layers. These layers generated time-dependent modifications in the ionic composition of both apical and basolateral solutions. Increases in sodium and chloride concentrations in the basolateral fluid were accompanied by reciprocal decreases in the concentrations of these ions in the apical fluid. The potassium concentration was increased in the apical, while decreased in the basolateral, solution. The total calcium concentration was slightly elevated in the apical, as compared with the basolateral fluid, while there were no alterations in pH. The endometrial layers demonstrated a significant transepithelial potential difference, and when this value was substituted in the Nernst equation a prediction of the passive distribution of ions across the cells was possible, indicating that none of the ions were in equilibrium. Addition of the sodium channel blocker amiloride to the medium bathing the cell layers reduced the modifications in ionic composition of apical and basolateral solutions. The data are consistent with other data indicating an amiloride-sensitive sodium-absorptive function for the endometrial epithelium. The ability of these primary cultures of endometrial epithelial cells to reduce the sodium while increasing the potassium concentration of the apical fluid is qualitatively in agreement with the low sodium and high potassium concentrations reported for human uterine fluid. The data suggest a role for the endometrial epithelium in generating and maintaining the distinctive ionic composition of the intra-uterine environment.