C-C chemokine receptor 5 (CCR5) is the primary coreceptor for human immunodeficiency virus type 1 (HIV-1) infection. Native chemokines that bind to CCR5 inhibit HIV-1 infection, albeit weakly, but chemically modified chemokines inhibit infection more efficiently. We have investigated the inhibitory mechanism of three N-terminally modified RANTES variants (AOP-, NNY-, and PSC-RANTES) with the MT-2 human T-cell line stably expressing either native or mutated CCR5. The RANTES analogues showed the same rank order (PSC > NNY > AOP) in their capacity to induce prolonged CCR5 internalization, inhibit surface reexpression, and prevent HIV-1 infection on MT-2 cells expressing wild-type CCR5 or CCR5 with four C-terminal serine phosphorylation sites mutated to alanine. None of the RANTES analogues caused internalization of a C-terminal cytoplasmic domain deletion mutant of CCR5, and each derivative had equal potency in inhibiting HIV-1 infection of MT-2 cells expressing this mutant. We conclude that the C-terminal cytoplasmic residues of CCR5 are necessary for receptor sequestration by RANTES analogues but that the process and the relative activity of each derivative are not dependent upon phosphorylation of the C-terminal serine residues. Two mechanisms of antiviral activity are demonstrated: receptor blockade and receptor sequestration. Potency correlates with the ability to induce CCR5 sequestration but not with receptor binding, suggesting that sequestration may make the greater contribution to antiviral activity.