Parathyroid hormone (PTH) inhibits proximal tubular brush border membrane Na+/Pi cotransport activity; this decrease in the transport activity was found to be associated with a decrease in type II Na+/Pi cotransporter protein content in rat brush border membranes. In the present study we investigated the PTH-dependent regulation of the type II Na+/Pi cotransporter in opossum kidney cells, a previously established model to study cellular mechanisms involved in the regulation of proximal tubular Na+/Pi cotransport. We transfected opossum kidney cells with a cDNA coding for NaPi-2 (rat renal type II Na+/Pi cotransporter). This allowed the study of PTH-dependent regulation of the transfected NaPi-2 and of the corresponding intrinsic cotransporter (NaPi-4). The results show (i) that the intrinsic and the transfected cotransporters are functionally (transport) and morphologically (immunofluorescence) localized at the apical membrane, (ii) that the intrinsic as well as the transfected Na+/Pi cotransport activities are inhibited by PTH, (iii) that PTH leads to a retrieval of both cotransporters from the apical membrane, (iv) that both cotransporters are rapidly degraded in response to PTH, and (v) that the reappearance/recovery of type II Na+/Pi cotransporter protein and function from PTH inhibition requires de novo protein synthesis. These results document that PTH leads to a removal of type II Na+/Pi cotransporters from the apical membrane and to their subsequent degradation.