The equilibrium state of CCR5 is manipulated here toward either activation or inactivation by introduction of single amino acid substitutions in the transmembrane domains (TMs) 6 and 7. Insertion of a steric hindrance mutation in the center of TM7 (G286F in position VII:09/7.42) resulted in biased signaling. Thus, β-arrestin recruitment was eliminated, whereas constitutive activity was observed in Gαi-mediated signaling. Furthermore, the CCR5 antagonist aplaviroc was converted to a full agonist (a so-called efficacy switch). Computational modeling revealed that the position of the 7TM receptor-conserved Trp in TM6 (Trp-248 in position VI:13/6.48, part of the CWXP motif) was influenced by the G286F mutation, causing Trp-248 to change orientation away from TM7. The essential role of Trp-248 in CCR5 activation was supported by complete inactivity of W248A-CCR5 despite maintaining chemokine binding. Furthermore, replacing Trp-248 with a smaller aromatic amino acid (Tyr/Phe) impaired the β-arrestin recruitment, yet with maintained G protein activity (biased signaling); also, here aplaviroc switched to a full agonist. Thus, the altered positioning of Trp-248, induced by G286F, led to a constraint of G protein active, but β-arrestin inactive and thus biased, CCR5 conformation. These results provide important information on the molecular interplay and impact of TM6 and TM7 for CCR5 activity, which may be extrapolated to other chemokine receptors and possibly to other 7TM receptors.