Apelin is a peptide involved in the regulation of body fluid homeostasis and cardiovascular functions, that was recently isolated as the endogenous ligand for the human orphan APJ receptor, a G protein-coupled receptor which shares 31% amino-acid sequence identity with the angiotensin II type 1 receptor. The predominant molecular forms of apelin naturally occuring in vivo are apelin 36, apelin 17 (K17F) and the pyroglutamyl form of apelin 13 (pE13F). We investigated the structure-activity relationships of apelin at the rat apelin receptor, tagged at its C-terminal end with enhanced green fluorescent protein and stably expressed in CHO cells. We compared the abilities of N- and C-terminal deleted fragments of K17F (KFRRQRPRLSHKGPMPF) to bind with high affinity to the apelin receptor, to inhibit cAMP production and to induce apelin receptor internalization. The first five N-terminal and the last two C-terminal amino acids of K17F were not essential for apelin binding or cAMP response. In contrast, deletion of the arginine in position 6 drastically decreased binding and cAMP response. The full-length sequence of K17F was the most potent inducer of apelin receptor internalization because successive N-terminal amino-acid deletions progressively reduced internalization and the removal of a single amino acid, the phenylalanine in position 17 at the C-terminus of K17F abolished this process. Thus, K16P binds with high affinity to the apelin receptor and strongly inhibits cAMP production, but does not induce apelin receptor endocytosis. These data indicate that apelin receptor signaling (coupling to Gi) and endocytosis are functionally dissociated, possibly reflecting the existence of several conformational states of this receptor, stabilized by the binding of different apelin fragments to the receptor. We then investigated the consequences for biological activity of this functional dissociation by evaluating the effects of various apelin fragments, injected iv, on arterial blood pressure in normotensive Wistar Kyoto rats. We showed that apelin fragments, that did not induce receptor internalization in vitro but kept their ability to activate receptor coupling to Gi, did not decrease arterial blood pressure. Our data showed that hypotensive actions of apelin peptides correlate with the ability of those ligands to internalize. Thus, the depressor response of apelin may be controlled by apelin receptor endocytosis, which is probably required for initiation of a second wave of signal transduction. The development of biaised agonists of the apelin receptor capable of promoting only one specific signal transduction pathway may therefore offer new therapeutic avenues for the treatment of cardiovascular disorders.