The present study was designed to examine the binding affinity and functional potency of selective angiotensin II type 1 (AT₁)-receptor antagonists towards specific mutants of AT₁ receptor using site-directed mutagenesis. We also compared our results with the wild-type AT₁ receptor and investigated the possible reasons behind that. Both wild-type and mutant receptors were expressed in COS-7 cells and the binding affinities of the antagonists were determined by radioligand binding assay. Inhibition of agonist-stimulated inositol phosphate accumulation by the antagonists was also done. Substitution of asparagine²³⁵ of intracellular loop 3 of the AT₁ receptor by arginine increased the binding affinity of the antagonists 5 – 34-fold, whereas the increase in the binding affinity of the antagonists in the phenylalanine²³⁹ mutant by arginine and tryptophan (F239R and F239W) were 3 – 19-fold and 2 – 15-fold higher, respectively, compared to the wild-type AT₁ receptor. The results suggested that substitution by a positively charged or sterically hindered amino acid in the AT₁ receptor allows it to interact with the acidic tetrazole moiety and carboxylate groups of the antagonists more strongly compared to the wild-type receptor. These findings may play an important role to change the binding affinity of the antagonists to an effective level for the pharmacological function of the drugs.