Electrical potential-driven 22Na+ fluxes were measured in membrane vesicles prepared from TBM-18(c123) cells (a clone of the established cell line TB-M). Fifty to seventy percent of the tracer uptake in vesicles derived from cells that were cultivated on a porous support were blocked by the diuretic amiloride. The amiloride inhibition constant was less than 0.1 microM, indicating that this flux is mediated by the apical Na+-specific channels. Vesicles prepared from cells that were not grown on a porous support exhibited much smaller amiloride-sensitive fluxes. Two Ca2+-dependent processes that down-regulate the channel conductance and were previously identified in native epithelia were found in the cultured cells as well. Vesicles isolated from cells that were preincubated with 5 X 10(-7) M aldosterone for 16-20 h exhibited higher amiloride-sensitive conductance than vesicles derived from control, steroid-depleted cells. Thus membrane derived from TBM-18(c123) cells can be used to characterize the epithelial Na+ channel and its hormonal regulation.