CXCL4L1, a platelet-derived ELR-negative CXC chemokine, is a powerful angiostatic and anti-tumoral chemokine. We developed a mass spectrometric assay for the detection of different natural CXCL4L1 isoforms. Using this assay, we identified 4 different CXCL4L1 isoforms in the supernatant of thrombin-stimulated platelets from healthy volunteers: the classical isoform CXCL4L1(1–70), CXCL4L1(-4–70), which probably arises through alternative signal peptide removal and two COOH-terminally truncated isoforms CXCL4L1(1–69) and CXCL4L1(-4–69). CXCL4L1(1–70) was the most abundant isoform, whereas CXCL4L1(-4–70) was detected in 50% of the platelet preparations. Since alterations to the NH2-terminus of chemokines can have severe biological consequences, we investigated the impact of the extension with 4 NH2-terminal amino acids on the biological activity of CXCL4L1. In vitro, CXCL4L1(-4–70) was as potent as CXCL4L1(1–70) in inhibiting signal transduction and migration of human microvascular endothelial cells towards vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2). In a FITC-conjugated dextran cell permeability assay, both splice variants showed a strong but comparable anti-permeable effect upon VEGF stimulation of the endothelial cell monolayer. In vivo angiogenesis induced by FGF-2 was equally reduced by CXCL4L1(1–70) and CXCL4L1(-4–70). In chemotaxis assays with CXCR3A-transfected cells the CXCL4L1 isoforms both induced migration from 125 ng/ml onward. Finally, CXCL4L1(1–70) and CXCL4L1(-4–70) showed the same affinity for heparin. In conclusion, the investigated biological activities of CXCL4L1 are not influenced by the four extra NH2-terminal residues present in the alternatively spliced isoform CXCL4L1(-4–70). Therefore, our results suggest that both isoforms equally interact with the CXCR3A and CXCR3B receptor.