Fyn is a member of the Src family of non-receptor-type tyrosine kinases and plays an important role in signal transductions regulating cell proliferation and differentiation. Fyn immunoreactivity was localized in the Sertoli cells of mouse testes. Although fyn-deficient adult male mice were fertile, a significant reduction in testis weight and degenerated germ cells were observed at 3 and 4 wk of age. Electron microscopic examination revealed that fyn −/− testis has ultrastructural abnormalities in the specialized junctional structures of the Sertoli cells, the ectoplasmic specializations. Unusual vesicular structures were found in the actin filament layers of the ectoplasmic specializations of mutant mice. Immunohistochemical studies demonstrated that both Fyn and actin filaments were concentrated in the areas of ectoplasmic specializations. At these sites, a high level of phosphotyrosine was also immunostained in wild-type testes, whereas phosphotyrosine immunoreactivity was reduced in fyn −/− testes. Immunoblot analyses revealed that Fyn was mainly distributed within the Triton X-100-insoluble cytoskeletal fraction prepared from wild-type testes, suggesting that Fyn might be associated with cytoskeletal proteins such as actin filaments. These findings suggest that Fyn kinase functions at the ectoplasmic specializations of the Sertoli cells in the testes, regulating the dynamics of cytoskeletal proteins. Fyn-mediated signal transduction in the Sertoli cells may affect the survival and differentiation of germ cells at a specific stage during spermatogenesis.