Objective To investigate the expression and localization of downstream signaling molecules of transforming growth factor beta (TGF-β) superfamily, Smad2 and Smad4 proteins, in postnatal cat testis, and to explore the function of Smad2 and Smad4 proteins in the regulation of testicular development and spermatogenesis. Methods Cellular localization of Smad2 and Smad4 proteins in juvenile, pubertal and sex maturity testes were examined by immunohistochemistry. Western blotting was performed to confirm that the anti-Smad2 and Smad4 antibodies would recognize specific proteins of the appropriate molecular weight in cat testis. BR> Results Smad2 and Smad4 mainly localizated in germ cells, Sertoli cells and Leydig cells of cat testis. The polyclone rabbit anti-Smad2 and Smad4 antibodies recognized a single band of about 58kD and 66kD in the cat testes, respectively. Conclusion Smad2 and Smad4 proteins are expressed in testicular cells during testicular development and spermatogenesis. It suggests that these two signaling molecules may play an important role in the regulation of cat testicular development and spermatogenesis. BR>