The mechanism by which the CXC chemokine platelet factor 4 (PF-4) inhibits endothelial cell proliferation is unclear. The heparin-binding domains of PF-4 have been reported to prevent vascular endothelial growth factor 165 (VEGF165) and fibroblast growth factor 2 (FGF2) from interacting with their receptors. However, other studies have suggested that PF-4 acts via heparin-binding independent interactions. Here, we compared the effects of PF-4 on the signalling events involved in the proliferation induced by VEGF165, which binds heparin, and by VEGF121, which does not. Activation of the VEGF receptor, KDR, and phospholipase Cγ (PLCγ) was unaffected in conditions in which PF-4 inhibited VEGF121-induced DNA synthesis. In contrast, VEGF165-induced phosphorylation of KDR and PLCγ was partially inhibited by PF-4. These observations are consistent with PF-4 affecting the binding of VEGF165, but not that of VEGF121, to KDR. PF-4 also strongly inhibited the VEGF165- and VEGF121-induced mitogen-activated protein (MAP) kinase signalling pathways comprising Raf1, MEK1/2 and ERK1/2: for VEGF165 it interacts directly or upstream from Raf1; for VEGF121, it acts downstream from PLCγ. Finally, the mechanism by which PF-4 may inhibit the endothelial cell proliferation induced by both VEGF121 and VEGF165, involving disruption of the MAP kinase signalling pathway downstream from KDR did not seem to involve CXCR3B activation.