The natural CC-chemokine RANTES(3–68), missing two NH₂-terminal residues, has been isolated from leukocytes and tumor cells. The highly specific aminopeptidase dipeptidyl peptidase IV (DPP IV), also called CD26, was shown to be responsible for this NH₂-terminal truncation of RANTES. Here it is reported that CD26/DPP IV treatment of RANTES enhances its anti-HIV-1 activity. RANTES(3–68) inhibited infection of PBMC by M-tropic HIV-1 strains ten-fold more efficiently than intact RANTES. This difference in antiviral potency between intact and truncated RANTES was even more pronounced (at least 25-fold) in CCR5-transfected cell lines. In HOS.CD4.CCR5 transfected cells, RANTES(1–68) had virtually no anti-HIV-1 activity (IC₅₀>130 nM), whereas RANTES(3–68) was a potent inhibitor of HIV-1 replication (IC₅₀: 5.5 nM). The anti-HIV-1 activity of RANTES(1–68) in the different cell types correlated with the expression of CD26. Moreover, the addition of soluble CD26 together with RANTES(1–68) significantly enhanced the antiviral activity of RANTES in HOS.CD4.CCR5 cells (IC₅₀: 13 nM). These observations point to an important role of CD26-mediated processing of RANTES in inhibiting the replication of CCR5-binding HIV strains in HIV-infected persons and in preventing the development of AIDS.