Vav is a signal transducing molecule containing C-terminal Src homology 3 (SH3)-SH2-SH3 domains, and has been thought to be expressed exclusively in hematopoietic and trophoblastic cells. By Northern blot analysis, vav transcripts of unique sizes, 4.8 kb and 1.0 kb, were detected in the testis among various tissues examined. From a mouse spermatocyte cDNA library, a novel isoform of vav (vav-T) was cloned, which corresponded to a part of the 4.8 kb transcript. Vav-T had an alternative 5' sequence up to the middle of SH2-coding region, and encoded 163 amino acids with a single SH3 domain. Northern blot analysis of fractionated testicular cells and in situ hybridization histochemistry demonstrated that vav-T transcripts were expressed in the differentiating germ cells, especially spermatocytes. A 24 kD protein was detected by anti-Vav antibodies in the testis, but not in the spleen or bone marrow. Transcripts of heterogeneous nuclear ribonucleoprotein K, known to associate with the most C-terminal SH3 domain of Vav, were also detected in the differentiating male germ cells. These results demonstrate expression of previously nondescribed Vav-isoform in the testicular germ cells, and suggest that it interacts with RNA-binding proteins and plays an important role in spermatogenesis.