PRO 2000 dose-dependently interfered with the binding of several anti-CD4 mAbs (clone OKT4, Leu3a, SK3) and anti-CXCR4 (clone 2B11 and 12G5) in PBMCs, whereas minor or no effects were observed on DC-SIGN and CCR5. The compound inhibited the CXCL12-induced signal transduction (IC50: 19.9 μg/ml), CXCR4 internalization (IC50: 9.5 μg/ml) and chemotaxis in PBMCs (IC50: 6.7 μg/ml). It inhibited CXCL12AF647 binding to T cells with an IC50 of 2.2 μg/ml. PRO 2000 did not induce signaling by itself. These CXCR4 antagonistic properties of PRO 2000 are potential additional mechanism of action that could explain the observation that PRO 2000 is more active against X4 viruses. In addition, we also examined the cellular activation potential and cytokines profile of PRO 2000 in PBMCs. PRO 2000 had minor effects on the induction of the activation markers CD25, CD69 and HLA-DR on T cells, but it did enhance the production of a small number of cytokines/chemokines, and most dramatically the production (~30-fold) of the specific CCR5 ligand MIP-1β.