To investigate the palmitoylation of the human bradykinin B2 receptor, we have mutated individually or simultaneously into glycine two potential acylation sites (cysteines 324 and 329) located in the carboxyl terminus of the receptor and evaluated the effects of these mutations by transfection in COS-7, CHO-K1, and HEK 293T. The wild-type receptor and the single mutants, but not the double mutant, incorporated [3H]palmitate, indicating that the receptor carboxyl tail can be palmitoylated at both sites. The mutants did not differ from the wild-type receptor for the kinetics of [3H]bradykinin binding, the basal and bradykinin-stimulated coupling to phospholipases C and A2, and agonist-induced phosphorylation. The nonpalmitoylated receptor had a 30% reduced capacity to internalize [3H]bradykinin. This indicates that palmitoylation does not influence the basal activity of the receptor and its agonist-driven activation. However, the mutants triggered phospholipid metabolism and MAP kinase activation in response to B2 receptor antagonists. Pseudopeptide and nonpeptide compounds that behaved as antagonists on the wild-type receptor became agonists on the nonpalmitoylated receptor and produced phospholipases C and A2 responses of 25-50% as compared to that of bradykinin. These results suggest that palmitoylation is required for the stabilization of the receptor-ligand complex in an uncoupled conformation.