The NY-ESO-1 gene, located on chromosome Xq28, encodes a cancer/testis antigen in human. Normally expressed only in germ cells, NY-ESO-1 is activated in a wide range of human tumors, eliciting both antibody and cell-mediated immune responses in cancer patients. Clinical immunotherapeutic trials NY-ESO-1 gene products are now ongoing. A closely related gene, LAGE-1, was subsequently identified and shares similar biological features. By database search, we have identified a third member of the human NY-ESO-1 gene family. This gene, designated ESO3, is also located on the X chromosome, clustered with two exact copies of ESO1(NY-ESO-1) and one copy of ESO2(LAGE-1), within a similar to400 kb segment. The exon-intron structures are conserved among ESO1-3. While ESO1 and ESO2 LAGE-1 share >80% protein sequence identity, homology between ESO3 and the other two members is lower (<50%). ES03 is also distinctive in that, unlike ESO1 and 2 that are normally expressed only in testis, ESO3 messenger RNA (mRNA) is ubiquitously expressed in somatic tissues. In addition to ES03, an intronless pseudogene highly homologous to ESO3 was found on chromosome 9, designated psiESO3. A search of the rodent databases identified mouse and rat counterparts of ESO3, named mESO3 and rESO3. mESO3 is similarly located on mouse X chromosome, with conserved exon-intron junctions. Protein sequence of mESO3 is 54% identical to ES03 (70% identical at the conserved carboxyl end), and 32% to ESO1 mESO3 mRNA is also ubiquitously expressed in somatic tissues, as is rESO3. In addition, an intronless and presumably non-coding, copy of the mESPO3, psimESO3, was identified on mouse chromosome 15. No counterpart of the ESO1 or 2 genes was found in rodents. (C) 2002 Elsevier Science B.V. All rights reserved.