Increased renal sodium retention is considered a major risk factor contributing to hypertension associated with chronic hyperinsulinemia and obesity. However, the molecular mechanism involved is not understood. Present study investigates the effect of insulin treatment on AT₁ receptor expression and angiotensin II (Ang II)-induced stimulation of Na/H-exchanger (NHE) and Na,K-ATPase (NKA) in OK cells, a proximal tubule cell line from opossum kidney. Presence of the AT₁ receptors in OK cells was confirmed by the specific binding of ¹²⁵I-sarangiotensin II and by detecting ~43 kDa protein on Western blot analysis with AT₁ receptor antibody and blocking peptide as well as by expression of AT₁ receptor mRNA as determined by RT-PCR. Insulin-treatment (100nM for 24hrs) caused an increase in ¹²⁵I-sar-angiotensin II binding, AT₁ receptor protein content and mRNA levels. The whole cell lysate and membrane showed similar insulin-induced increase in the AT₁ receptor protein expression, which was blocked by genistein (100 nM), a tyrosine kinase inhibitor and cycloheximide (1.5µg/mL), a protein synthesis inhibitor. Determination of ethyl isopropyl amiloride sensitive ²²Na⁺-uptake, a measure of the NHE activity, revealed that Ang II (1-100 pM)-induced stimulation of NHE in insulin-treated cells was significantly greater than in the control cells. Similarly, Ang II (1-100 pM)-induced stimulation of ouabain-sensitive ⁸⁶Rb⁺-uptake, a measure of NKA activity, in insulin-treated cells was significantly greater than in the control cells. Ang II stimulation of both the transporters was blocked by AT₁ receptor antagonist losartan, suggesting the involvement of AT₁ receptors. Thus, chronic insulin treatment causes up-regulation of AT₁ receptors, which evoked Ang II-induced stimulation of NHE and NKA. We propose that insulin-induced increase in the renal AT₁ receptor function serves as a mechanism responsible for the increased renal sodium reabsorption and thus may contribute to development of hypertension in conditions associated with hyperinsulinemia.