During water deprivation, prostaglandin E₂ (PGE₂), formed by renal medullary interstitial cells (RMICs), feedback-inhibits the actions of antidiuretic hormone. Interstitial PGE₂ concentrations represent the net of both PGE₂ synthesis by cyclooxygenase (COX) and PGE₂ uptake by carriers such as PGT. We used cultured RMICs to examine the effects of hyperosmolarity on both PG synthesis and PG uptake in the same RMIC cells. RMICs expressed endogenous PGT as assessed by mRNA and immunoblotting. RMICs rapidly took up ³H-PGE₂ to a level 5-10-fold above background and with a characteristic time-dependent "overshoot". Inhibitory constants (K_i) for various PGs and PGT inhibitors were similar between RMICs and the cloned rat PGT. Increasing extracellular hyperosmolarity to the range of 335-485 mOsm/kg increased the net release of PGE₂ by RMICs, an effect that was concentration-dependent, maximal by 24 hours, reversible, and associated with increased expression of COX-2. Over the same time period, there was decreased cell-surface activity of PGT due to internalization of the transporter. With continued exposure to hyperosmolarity over 7-10 days, PGE₂ release remained elevated, COX-2 returned to baseline, and PGT-mediated uptake became markedly reduced. Our findings suggest that hyperosmolarity induces coordinated changes in COX-2 mediated PGE₂ synthesis and PGT-mediated PGE₂ uptake in RMICs.