Chemokine receptor CXCR4 is involved in tumor growth, angiogenesis and metastasis. Its function is regulated in many ways and one of them is alternative splicing. We identified two novel coding splice variants (CXCR4-3 and CXCR4-4) of CXCR4 in Ewing sarcoma (EWS) cell lines by whole transcriptome sequencing and validated these with reverse transcriptase-PCR and Sanger sequencing. The novel splice variants were expressed at RNA level in Ewing sarcoma samples and in other tumor cell lines and placenta, but not in lung. Due to inclusion of an additional exon the new isoforms have a 70 and 33 amino acid elongation of the N-terminal end of CXCR4. For validation at protein and functional level, the identified isoforms and normal CXCR4 were cloned into an EYFP tagged vector and ectopically expressed in HEK293T cell line and EWS cell line A673. Of the novel isoforms CXCR4-3 showed cell membrane localization and a functional response after addition of CXCR4 ligand CXCL12a. CXCR4-4 showed strong cytoplasmic accumulation and no response to ligand treatment. The role of the newly discovered isoforms in CXCR4 signaling is likely to be limited. Our data stresses the importance of functional validation of newly identified isoforms. (C) 2015 Elsevier Inc. All rights reserved.