A reconstructed monolayer was formed using epithelial cells from normal mouse kidney to investigate the hormonal effect on phosphate transport by the renal cells. The cells, when cultured on a Millipore filter, formed a monolayer with an apical negative transepithelial potential of 8.4 +/- 0.4 mV. When radioactive phosphate was added onto the apical surface of the monolayer (corresponding to the luminal surface of a renal tubule), the phosphate was transported through the cell layer to the basolateral surface (corresponding to the peritubular surface of a renal tubule). This transport process was saturable, energy-dependent, and inhibited by 2,4-dinitrophenol or ouabain. Dose-dependent parathyroid hormone-induced inhibition (73% of the control) was also evident in this system. Similar inhibition (69% of the control) was observed with DBcAMP. Thus, monolayers reconstructed from cultured mouse kidney cells show characteristics similar to those of renal tubules.