Parathyroid hormone (PTH) produces rapid inhibition of Na(+)-phosphate cotransport, characterized by a decreased maximal rate of transport, and the inhibition is independent of de novo protein synthesis. The present study determined whether the action of PTH on Na(+)-phosphate cotransport is mediated, at least in part, by rapid endocytic internalization of Na(+)-phosphate cotransporters present in the plasma membrane. Horseradish peroxidase, a fluid-phase marker, was used to demonstrate the presence of endocytosis in opossum kidney (OK) epithelial cells in monolayer culture. An increase in medium osmolality to 500 mosmol/kgH2O, by addition of sucrose, produced 80% inhibition of endocytosis within 1 h. The inhibition was reversed on returning the cells to normal medium. Incubation of OK cell monolayers with PTH (10(-8) M) for 3 h at normal osmolality (281 mosmol/kgH2O) inhibited Na(+)-phosphate cotransport (4 min uptakes) by 56-67%. In hyperosmolar medium (513 mosmol/kgH2O), when endocytosis was inhibited, PTH inhibited Na(+)-phosphate cotransport by only 25-39%, a change that was significantly different from the inhibition in normal medium. Hyperosomolality had no effect on PTH inhibition of Na(+)-H+ exchange or on PTH stimulation of intracellular adenosine 3',5'-cyclic monophosphate. We conclude that the full inhibitory action of PTH on Na(+)-phosphate cotransport may require an intact endocytic mechanism.