Mtsarg1 (Mus musculus testis and spermatogenesis cell apoptosis-related gene 1) gene with 1103 bp in full length had been cloned previously (GenBank accession number: AF399971, 2002; re-designated as Spata3, Mus musculus spermatogenesis-associated 3, 2007). In the present study, we identified a novel transcript variant of Mtsarg1, named Mtsarg1-beta which is 887 bp in length (GenBank accession numbers: EU259321 and EF546784, 2007, designated as Spata3 variant 4) by reverse transcription-polymerase chain reaction (RT-PCR), cloning and sequencing. Mtsarg1-beta which has high similarity with Mtsarg1 contains an entire open reading frame of 417 bp encoding a protein consisting of 138 amino acids. Mtsarg1-beta protein is a non-secretory protein with a theoretic molecular mass around 14.79 kD and an isoelectric point of 9.74, which shares the 100 N-terminal amino acids with Mtsarg1 followed by 38 amino acids differing from Mtsarg1. Multi-tissue RT-PCR results and Northern blot analysis for adult DBA/2 mice showed that Mtsarg1-beta and Mtsarg1 mRNAs were specifically expressed in testis at high level. RT-PCR results also showed that Mtsarg1-beta and Mtsarg1 mRNAs were not expressed in mouse GC-1 spermatogonia. In situ hybridization revealed that both Mtsarg1 and probably Mtsarg1-beta mRNAs were mainly expressed in mouse spermatocytes. Subcellular localization analysis suggested that Mtsarg1 protein was mainly localized in nucleus while Mtsarg1-beta protein was mainly localized in cytoplasm. All these results indicate that Mtsarg1 and Mtsarg1-beta may play an important role in mouse testicular function and in spermatocyte development.