Using the digital differential display program of the National Center for Biotechnology Information, we identified a contig of expression sequence tags (ESTs) (Accession No. BM316936), which came from zebrafish ovary and testis libraries. The full-length cDNA of this transcript was cloned and further confirmed by polymerase chain reaction and sequencing. The full-length cDNA of the novel gene is 807 bp and encodes a novel protein of 187 amino acids, which shares no significant homology with any other known proteins. Characterization of genomic sequences of the gene revealed that it spans 6 kb on the linkage group 3 and is composed of five exons and four introns. RT-PCR analysis showed that it was expressed in mature oocytes and one-cell stage, and persisted until 24 h of development. RT-PCR also revealed that it is expressed in gonad and kidney, with the highest level of expression in the testis. The expression sites of the novel gene in adult gonad were further localized by in situ hybridization to oogonia and growing oocytes in ovary and to spermatogonia, spermatocytes but not to spermatids in testis. Based on its abundance in testis and the germline stem cell - spermatogonia and oogonia, we hypothesize that it may function as a testicular development and gametogenesis related gene that plays important roles in spermatogenesis, and named it Zsrg (zebrafish testis spermatogenesis related gene, Zsrg). (c) 2005 Elsevier Inc. All rights reserved.