Because SDF-1/54 targets the co-receptor CXCR4, which is essential for viral entry, we validated its role as an HIV-1 entry inhibitor by a time-of-addition assay in which we studied the inhibitory activity of SDF-1/54 against CXCR4-tropic HIV-1 IIIB when it was added to cells at different intervals post-infection. Here, CXCL12 is linked to infection.