Previously, we showed that angiotensin II (Ang II) receptors in cultured rat renomedullary interstitial cells (RMICs) are osmotically regulated (1). The current study examined the mechanisms underlying this osmotic regulation in RMICs cultured in isoosmotic (300 mOsm/kg H₂O) and hyperosmotic (600 mOsm/kg H₂O) conditions. Radioligand competition analysis coupled with RNase protection assays (RPA) and ligand-mediated receptor internalization studies revealed that RMICs primarily express the type 1a angiotensin receptor (AT_1aR). When cultured under hyperosmotic conditions, the density (B_max) of AT₁R in RMIC membranes decreased by 31% [B_max (pmol/mg protein): 300 mOsm/kg H₂O, 6.44 ±0.46 vs 600 mOsm/kg H₂O, 4.42 ±0.37, n=8, P<0.01], under conditions in which no detectable changes in AT_1aR mRNA expression or in the kinetics of ligand-mediated AT₁R internalization were observed. RNA electromobility shift assays showed that RNA-protein complex (RPC) formation between RMIC cytosolic RNA binding proteins and the 5' leader sequence (5'LS) of the AT_1aR was increased 1.5-fold under hyperosmotic conditions [5'LS RPC (arbitrary units): 300 mOsm/kg H₂O, 0.79 ±0.08 vs 600 mOsm/kg H₂O, 1.17 ±0.07; n=4, P<0.01]. These results suggest that the downregulation of AT_1aR expression in RMICs cultured under hyperosmotic conditions is regulated at the post-transcriptional level by RNA binding proteins that interact within the 5'LS of the AT_1aR mRNA. The downregulation of AT_1aR expression under hyperosmotic conditions may be an important mechanism by which the activity of Ang II is regulated in the hyperosmotic renal medulla.