Prostaglandin E2 (PGE2) is known to inhibit transepithelial Cl transport in medullary thick ascending limb (mTAL), but the mechanism of inhibition or the transport pathway affected has not been identified. We undertook this study to examine the effect of PGE2 on Na-K-2Cl cotransport in mouse mTAL cells in culture. In nanomolar concentrations, PGE2 inhibited the Na- and Cl-dependent, bumetanide-sensitive K influx by 45%, and this inhibition was also observed in the presence of 3 mM ouabain. Although PGE2 also inhibited ouabain-sensitive K flux, that inhibition was abolished in the presence of apical nystatin, suggesting that the pump inhibition was secondary to diminished Na entry into the cells. The effect of PGE2 was concentration dependent. Inhibition was observed at a concentration of < 1 nM, and half-maximal effect was observed at 2.5 nM. The effect of PGE2 was not mediated by an action on cytosolic Ca because cytosolic Ca was unchanged after the addition of PGE2. PGE2 reduced the maximal velocity for the cotransporter but had no effect on the affinity of the cotransporter for external Na, K, or Cl. Specific [3H]bumetanide binding was reduced in the presence of PGE2, suggesting that PGE2 affected bumetanide-sensitive K influx by downregulating the number of functioning Na-K-2Cl cotransporters. These results suggest that Na-K-2Cl cotransport in the mTAL cells may be under tonic inhibitory control of PGE2.